Scaffolds for cell transplantation

ABSTRACT

A device that includes a scaffold composition and a bioactive composition with the bioactive composition being incorporated into or coated onto the scaffold composition such that the scaffold composition and/or a bioactive composition controls egress of a resident cell or progeny thereof. The devices mediate active recruitment, modification, and release of host cells from the material.

GOVERNMENT SUPPORT

The invention was supported, in whole, or in part, by NIH/NICDR grantnumber RO1DE13349 and HL069957. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

The total costs of musculoskeletal disease in the US in 2000 have beenestimated at US $ 254 billion and, in developing countries, the figureis estimated at US$ 100 billion. Under normal conditions, muscles canrepair themselves by regenerating damaged muscle fibers and restoringmuscle strength. Following an initial necrosis of damaged muscle fibers,an inflammatory response is initiated that activates a residentpopulation of quiescent cells termed satellite cells. These myogeniccells proliferate, migrate to the site of injury, differentiate and fuseto form mature myofibers, or fuse with existing myofibers thusregenerating damaged muscle fibers and restoring their function. Whenthese normal processes are compromised by disease or age, damaged musclefibers are instead replaced by infiltrating fibrous tissue or fat,leading to a net loss of muscle mass and a resultant loss of strength.

Cell transplantation has been used in regenerative medicine formusculoskeletal disorders as well as degenerative conditions such asdiabetes with limited success. Limitations of earlier approaches includeloss of cell viability and function following transplantation.

SUMMARY OF THE INVENTION

The devices and methods of the invention provide a solution to severalproblems associated with previous cell transplantation protocols.Transplantation systems that enhance the viability of the cells andinduce their outward migration to populate injured or defective bodilytissues enhance the success of tissue regeneration, e.g., theregeneration of muscle tissue or other tissues as well as angiogenesis.Such a device that controls cell function and/or behavior, e.g.,locomotion, contains a scaffold composition and one or more bioactivecompositions. The bioactive composition is incorporated into or coatedonto the scaffold composition. The scaffold composition and/or bioactivecomposition temporally and spatially (directionally) controls egress ofa resident cell or progeny thereof.

The devices mediate active recruitment, modification, and release ofhost cells from the material in vivo, thereby improving the function ofcells that have resided in the scaffold. For example, the deviceattracts or recruits cells already resident in the body to the scaffoldmaterial, and programs or reprograms the resident cells to a desiredfate (e.g., immune activation or tissue regeneration).

This device includes a scaffold composition which incorporates or iscoated with a bioactive composition; the device regulates the egress ofresident cells. Egress is regulated spatially and temporally. Dependingon the application for which the device is designed, the deviceregulates egress through the physical or chemical characteristics of thescaffold itself. For example, the scaffold composition is differentiallypermeable, allowing cell egress only in certain physical areas of thescaffold. The permeability of the scaffold composition is regulated, forexample, by selecting or engineering a material for greater or smallerpore size, density, polymer cross-linking, stiffness, toughness,ductility, or viscoelasticity. The scaffold composition containsphysical channels or paths through which cells can move more easilytowards a targeted area of egress of the device or of a compartmentwithin the device. The scaffold composition is optionally organized intocompartments or layers, each with a different permeability, so that thetime required for a cell to move through the device is precisely andpredictably controlled. Migration is also regulated by the degradation,de- or re-hydration, oxygenation, chemical or pH alteration, or ongoingself-assembly of the scaffold composition. These processes are driven bydiffusion or cell-secretion of enzymes or other reactive chemicals.

Alternatively or in addition, egress is regulated by a bioactivecomposition. By varying the concentration of growth factors,homing/migration factors, morphogens, differentiation factors,oligonucleotides, hormones, neurotransmitters, neurotransmitter orgrowth factor receptors, interferons, interleukins, chemokines,cytokines, colony stimulating factors, chemotactic factors,extracellular matrix components, adhesion molecules and other bioactivecompounds in different areas of the device. The device controls anddirects the migration of cells through its structure. Chemicalaffinities are used to channel cells towards a specific area of egress.For example, adhesion molecules are used to attract or retard themigration of cells. By varying the density and mixture of thosebioactive substances, the device controls the timing of the migrationand egress. The density and mixture of these bioactive substances iscontrolled by initial doping levels or concentration gradient of thesubstance, by embedding the bioactive substances in scaffold materialwith a known leaching rate, by release as the scaffold materialdegrades, by diffusion from an area of concentration, by interaction ofprecursor chemicals diffusing into an area, or by production/excretionof compositions by resident support cells. The physical or chemicalstructure of the scaffold also regulates the diffusion of bioactiveagents through the device.

The bioactive composition includes one or more compounds that regulatecell function and/or behavior. The bioactive composition is covalentlylinked to the scaffold composition or non-covalently associated with thescaffold. For example, the bioactive composition is an extracellularmatrix (ECM) component that is chemically crosslinked to the scaffoldcomposition. Regardless of the tissue of origin, ECM componentsgenerally include three general classes of macromolecules: collagens,proteoglycans/glycosaminoglycans (PG/GAG), and glycoproteins, e.g.,fibronectin (FN), laminin, and thrombospondin. ECM components associatewith molecules on the cell surface and mediate adhesion and/or motility.Preferably, the ECM component associated with the scaffold is aproteoglycan attachment peptide or cyclic peptide containing the aminoacid sequence arginine-glycine-aspartic acid (RGD). Proteoglycanattachment peptides are selected from the group consisting of G₄RGDSP(SEQ ID NO: 1), XBBXBX (SEQ ID NO: 2), PRRARV (SEQ ID NO: 3),YEKPGSPPREVVPRPRPGV (SEQ ID NO:4), RPSLAKKQRFRHRNRKGYRSQRGHSRGR (SEQ IDNO: 5), and RIQNLLKITNLRIKFVK (SEQ ID NO: 6), and cell attachmentpeptides are selected from the group consisting of RGD, RGDS, LDV, REDV,RGDV, LRGDN (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), YIGSR (SEQ ID NO: 9),PDSGR (SEQ ID NO: 10), RNIAEIIKDA (SEQ ID NO: 11), RGDT, DGEA, and VTXG.

Components of the ECM, e.g., FN, laminin, and collagen, interact withthe cell surface via the integrin family of receptors, a group ofdivalent cation-dependent cell surface glycoproteins that mediatecellular recognition and adhesion to components of the ECM and to othercells. Ligands recognized by integrins typically contain an RGD aminoacid sequence that is expressed in many ECM proteins. Exemplarymolecules that mediate cell adhesion and/or movement include FN,laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin,aggrecan, agrin, bone sialoprotein, cartilage matrix protein,fibronogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen,restrictin, serglycin, SPARC/osteonectin, versican, von WillebrandFactor, polysacchride heparin sulfate, cell adhesion molecules includingconnexins, selectinsinclude collagen, RGD (Arg-Gly-Asp) and YIGSR(Tyr-Ile-Gly-Ser-Arg) peptides, glycosaminoglycans (GAGs), hyaluronicacid (HA), integrins, selectins, cadherins and members of theimmunoglobulin superfamily. Carbohydrate ligands of the ECM include thepolysaccharides hyaluronic acid, and chondroitin-6-sulfate.

Signal transduction events that participate in the process of cellmotility are initiated in response to cell growth and/or celldifferentiation factors. Thus, the device optionally contains a secondbioactive composition that is a growth factor, morphogen,differentiation factor, or chemoattractant. For example, the deviceincludes vascular endothelial growth factor (VEGF), hepatocyte growthfactor (HGF), or fibroblast growth factor 2 (FGF2) or a combinationthereof. Other factors include hormones, neurotransmitters,neurotransmitter or growth factor receptors, interferons, interleukins,chemokines, MMP-sensitive substrate, cytokines, colony stimulatingfactors. Growth factors used to promote angiogenesis, bone regeneration,wound healing, and other aspects of tissue regeneration are listedherein and are used alone or in combination to induce colonization orregeneration of bodily tissues by cells that have migrated out of animplanted scaffold device.

Alternatively, the second bioactive composition is an inhibitor ofdifferentiation. In this case, the cells are maintained at a desiredstage of development or differentiation by the inhibitor until theinhibitor is depleted (e.g., has diffused out of the scaffold or isrendered inactive) or until another signal is provided to induce achange, e.g., to promote maturation or differentiation.

In some cases, the second bioactive composition is covalently linked tothe scaffold composition, keeping the composition relatively immobilizedin or on the scaffold composition. In other cases, the second bioactivecomposition is noncovalently associated with the scaffold. Noncovalentbonds are generally one to three orders of magnitude weaker thancovalent bonds permitting diffusion of the factor out of the scaffoldand into surrounding tissues. Noncovalent bonds include electrostatic,hydrogen, van der Waals, π aromatic, and hydrophobic. For example, agrowth factor such as VEGF is associated with the device by noncovalentbonds and exits the device following administration of the cell-seededdevice to a target site to further promote angiogenesis and tissuerepair of the target bodily tissue.

The scaffold composition is biocompatible. The composition isbio-degradable/erodable or resistant to breakdown in the body.Relatively permanent (degradation resistant) scaffold compositionsinclude metals and some polymers such as silk. Preferably, the scaffoldcomposition degrades at a predetermined rate based on a physicalparameter selected from the group consisting of temperature, pH,hydration status, and porosity, the cross-link density, type, andchemistry or the susceptibility of main chain linkages to degradation orit degrades at a predetermined rate based on a ratio of chemicalpolymers. For example, a high molecular weight polymer comprised ofsolely lactide degrades over a period of years, e.g., 1-2 years, while alow molecular weight polymer comprised of a 50:50 mixture of lactide andglycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, 10 weeks.A calcium cross-linked gels composed of high molecular weight, highguluronic acid alginate degrade over several months (1, 2, 4, 6, 8, 10,12 months) to years (1, 2, 5 years) in vivo, while a gel comprised oflow molecular weight alginate, and/or alginate that has been partiallyoxidized, will degrade in a matter of weeks.

In one example, cells mediate degradation of the scaffold matrix, i.e.,the scaffold composition is enzymatically digested by a compositionelicited by a resident cell, and the egress of the cell is dependentupon the rate of enzymatic digestion of the scaffold. In this case,polymer main chains or cross-links contain compositions, e.g.,oligopeptides, that are substrates for collagenase or plasmin, or otherenzymes produced by within or adjacent to the scaffold.

Exemplary scaffold compositions include polylactic acid, polyglycolicacid, PLGA polymers, alginates and alginate derivatives, gelatin,collagen, fibrin, hyaluronic acid, laminin rich gels, agarose, naturaland synthetic polysaccharides, polyamino acids, polypeptides,polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohols),poly(alkylene oxides), poly(allylamines)(PAM), poly(acrylates), modifiedstyrene polymers, pluronic polyols, polyoxamers, poly(uronic acids),poly(vinylpyrrolidone) and copolymers or graft copolymers of any of theabove. One preferred scaffold composition includes an RGD-modifiedalginate.

Porosity of the scaffold composition influences egress of the cells fromthe device. Pores are nanoporous, microporous, or macroporous. Forexample, the diameter of nanopores are less than about 10 nm; microporeare in the range of about 100 nm-20 μm in diameter; and, macropores aregreater than about 20 μm (preferably greater than about 100 μm and evenmore preferably greater than about 400 μm). In one example, the scaffoldis macroporous with aligned pores of about 400-500 μm in diameter.

The devices are manufactured in their entirety in the absence of cellsor can be assembled around or in contact with cells (the material isgelled or assembled around cells in vitro or in vivo in the presence ofcells and tissues) and then contacted with cells to produce acell-seeded structure. Alternatively, the device is manufactured in twoor more (3, 4, 5, 6, . . . 10 or more) stages in which one layer orcompartment is made and seeded with cells followed by the constructionof a second, third, fourth or more layers, which are in turn seeded withcells in sequence. Each layer or compartment is identical to the othersor distinguished from one another by the number, genotype, or phenotypeof the seed cell population as well as distinct chemical, physical andbiological properties. Prior to implantation, the device is contactedwith purified populations cells or characterized mixtures of cells asdescribed above. Exemplary cells include myoblasts for muscleregeneration, repair or replacement; hepatocytes for liver tissueregeneration, repair or organ transplantation, chondrocytes forcartilage replacement, regeneration or repair, and osteoblasts for boneregeneration, replacement or repair, various stem cell populations(embryonic stem cells differentiated into various cell types), bonemarrow or adipose tissue derived adult stem cells, cardiac stem cells,pancreatic stem cells, endothelial progenitors and outgrowth endothelialcells, mesenchymal stem cells, hematopoietic stem cells, neural stemcells, satellite cells, side population cells, differentiated cellpopulations including osteoprogenitors and osteoblasts, chondrocytes,keratinocytes for skin, tenocytes for tendon, intestinal epithelialcells, endothelial cells, smooth muscle cells and fibroblasts for tissueor organ regeneration, repair or replacement and/or for DNA delivery.Preferably, the cells are human; however, the system is adaptable toother eucaryotic animal cells, e.g., canine, feline, equine, bovine, andporcine as well as prokaryotic cells such as bacterial cells.

A method of making a scaffold is carried out by providing a scaffoldcomposition and covalently linking or noncovalently associating thescaffold composition with a first bioactive composition. The firstbioactive composition preferably contains a cell adhesion ligand. Thescaffold composition is also contacted with a second bioactivecomposition. The second bioactive composition is preferablynon-covalently associated with the scaffold composition to yield a dopedscaffold, i.e., a scaffold composition that includes one or morebioactive substances. The contacting steps are optionally repeated toyield a plurality of doped scaffolds, e.g., each of the contacting stepsis characterized by a different amount of the second bioactivecomposition to yield a gradient of the second bioactive composition inthe scaffold device. Rather than altering the amount of composition,subsequent contacting steps involve a different bioactive composition,i.e., a third, fourth, fifth, sixth . . . , composition or mixture ofcompositions, that is distinguished from the prior compositions ormixtures of prior doping steps by the structure or chemical formula ofthe factor(s). The method optionally involves adhering individualniches, layers, or components to one another and/or insertion ofsemi-permeable, permeable, or nonpermeable membranes within or at one ormore boundaries of the device to further control/regulate locomotion ofcells or bioactive compositions. As described above, the scaffold isseeded with cells after completion of the construction of the device orin an iterative manner throughout the construction of each component.

Therapeutic applications of the device include tissue generation,regeneration/repair, as well as augmentation of function of a mammalianbodily tissue, and the targeted destruction of undesired tissues (e.g.,cancer, undesired adipose depots), as well as the instruction of immunecells. For example, the method includes the steps of providing a devicethat includes scaffold composition with a bioactive compositionincorporated therein or thereon and a mammalian cell bound to thescaffold. A mammalian tissue is contacted with the device. The scaffoldcomposition temporally controls egress of the cell and the bioactivecomposition spatially or directionally regulates egress of the cell. Inanother example, the device that is provided contains a scaffoldcomposition with a bioactive composition incorporated therein or thereonand a mammalian cell immobilized within the scaffold. In the lattercase, the cell remains immobilized within the scaffold, and the scaffoldcomposition temporally controls egress of a progeny cell of theimmobilized cell and the bioactive composition spatially regulatesegress of the progeny cells.

A method of modulating an activity of a cell, e.g., a host cell, iscarried out by administering to a mammal a device containing a scaffoldcomposition and a recruitment composition incorporated therein orthereon, and then contacting the cell with a deployment signal. Thedeployment signal induces egress of the cells from the device. Theactivity of the cell at egress differs from that prior to entering thedevice. Cells are recruited into the device and remain resident in thedevice for a period of time, e.g., minutes; 0.2. 0.5, 1, 2, 4, 6, 12, 24hours; 2, 4, 6, days; weeks (1-4), months (2, 4, 6, 8, 10, 12) or years,during which the cells are exposed to structural elements and bioactivecompositions that lead to a change in the activity or level of activityof the cells. The cells are contacted with or exposed to a deploymentsignal that induces induces egress of the altered (re-educated orreprogrammed) cells and the cells migrate out of the device and intosurrounding tissues or remote target locations.

The deployment signal is a composition such as protein, peptide, ornucleic acid. For example, cells migrating into the device onlyencounter the deployment signal once they have entered the device. Insome cases, the deployment signal is a nucleic acid molecule, e.g., aplasmid containing sequence encoding a protein that induces migration ofthe cell out of the device and into surrounding tissues. The deploymentsignal occurs when the cell encounters the plasmid in the device, theDNA becomes internalized in the cell (i.e., the cell is transfected),and the cell manufactures the gene product encoded by the DNA. In somecases, the molecule that signals deployment is an element of the deviceand is released from the device in delayed manner (e.g., temporally orspatially) relative to exposure of the cell to the recruitmentcomposition. Alternatively, the deployment signal is a reduction in orabsence of the recruitment composition. For example, a recruitmentcomposition induces migration of cells into the device, and a reductionin the concentration or depletion, dissipation, or diffusion of therecruitment composition from the device results in egress of cells outof the device. In this manner, immune cells such as T cells, B cells, ordendritic cells (DCs) of an individual are recruited into the device,primed and activated to mount an immune response against anantigen-specific target. Optionally, an antigen corresponding to atarget to which an immune response is desired is incorporated into oronto the scaffold structure. Cytokines, such as granulocyte macrophagecolony stimulating factor (GM-CSF) are also a component of the device toamplify immune activation and/or induce migration of the primed cells tolymph nodes. Other cell specific recruitment compositions are describedbelow. For example, vascular endothelial growth factor (VEGF) is usefulto recruit angiogenic cells.

The device recruit cells in vivo, modifies these cells, and thenpromotes their migration to another site in the body. This approach isexamplied herein in the context of dendritic cells and cancer vaccinedevelopment but is also useful to other vaccines such as those againstmicrobial pathogens as well as cell therapies in general. Cells educatedusing the devices described herein promote regeneration of a tissue ororgan immediately adjacent to the material, or at some distant site.Alternatively, the cells are educated to promote destruction of a tissue(locally or at a distant site). The methods are also useful for diseaseprevention, e.g., to promote cell-based maintenance of tissue structureand function to stop or retard disease progression or age-related tissuechanges. The education of cells within the device, “programming” and“reprogramming” permits modification of the function or activity of anycell in the body to become a multipotent stem cell again and exerttherapeutic effects.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. All references cited herein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photomicrographs showing that the purity of primaryskeletal muscle myoblast cultures is increased by Percoll fractionation.FIG. 1A shows an initial isolation resulted in heterogeneous cellpopulation with myogenic cells (arrows) staining for both desmin andHoescht nuclear stain. In contrast non-myogenic cells are only stainedwith the nuclear dye (arrow heads). FIG. 1B shows that Percollfractionation resulted in homogeneous myogenic cultures.

FIG. 2A is a line graph showing HGF release from macroporous, nonpeptide modified alginate scaffolds, values represent mean and standarddeviation (n=5). FIG. 2B is a bar graph showing Viability and FIG. 2C isa bar graph showing cumulative migration of primary myoblasts, in andfrom respectively, nanoporous scaffolds (▪), nanoporous scaffoldsreleasing HGF (

), and microporous scaffolds releasing HGF (

). Values represent mean and SD (n=6).

FIGS. 3A and 3B are bar graphs showing cell condition and performance.Viability (FIG. 3A) and cumulative migration (FIG. 3B) is enhanced bypeptide modification of alginate. Bars represent nanoporous alginatescaffolds (▪), nanoporous scaffolds with HGFG release (

), and microporous scaffolds with HGF release (

). Numbers represent mean and SD (n=8). * indicates statisticallysignificant difference as compared to non-HGFG releasing scaffoldsp<0.001.

FIG. 4A is a scanning electron microscope (SEM) photomicrographs of sideand end views of peptide modified macroporous alginate and FIG. 4B is aline graph showing release kinetics of FGF2. Values represent mean andSD (n=4).

FIG. 5A is a bar graph showing Cell viability and FIG. 5B is a bar graphshowing that migration is enhanced by macroporosity and FGF2 releasefrom peptide modified algnate scaffolds. Bars represent macroporousalginate (▪), macroporous alginate scaffolds releasing HGF (

), and scaffolds releasing both HGF and FGF2 (

). Values represent mean values and SD (n=6). * indicates statisticallysignificant differences (p<0.01), as compared to non-HGF releasingconditions.

FIG. 6 is a photograph of a Western Blot analysis of cell lysates frommyoblasts cultured in macroporous scaffolds fabricated from peptidemodified alginate either with (+) (Lane 2) or without (−) (Lane 3)release of HGF and FGF2. Cells migrating from scaffolds were alsoanalyzed (Lane 4). Controls of lysates from C2Cl2 cell line (Lane 1),and primary myoblasts cultured in standard two dimensional conditionsthat promote differentiation (Lane 5) are also shown.

FIGS. 7A-7C are photographs of tibialis anterior muscles treated withscaffolds delivering cells and releasing HGF and FGF2 (FIG. 7A),scaffolds containing only HGF and FGF2 (FIG. 7B), and scaffoldscontaining only myoblasts (FIG. 7C). Muscles were stained to allow grossidentification of regions containing lac Z donor cells (dotted linesoutline positively stained tissue). Size bars are shown on thephotomicrographs. FIG. 7D is a bar graph showing that the mass of themuscle at 30 days post injury was greater when treated with scaffoldscontaining myoblasts and HGF and FGF2 (HGF/FGF2-cells in scaffold)compared to injuries treated with an injection of myoblasts directlyinto the muscle (cells[injected]), blank scaffolds, scaffolds releasinggrowth factors without cells (HGF/FGF), or cells transplanted inscaffolds not releasing growth factors (cells in scaffold). Valuesrepresent mean and standard deviation (n=6). * represents statisticallysignificant difference (p<0.001) compared to all other conditions.

FIGS. 8A-J are photomicrographs of defects 10 days post injury (FIGS.8A-E), and defects 30 days post injury (FIGS. 8F-J). Conditions includedinjuries treated with an injection of myoblasts directly into the muscle(FIGS. 8A, F), blank scaffolds (FIGS. 8B, G), scaffolds releasing growthfactors without cells (FIGS. C, H), cells transplanted in scaffolds notreleasing growth factors (FIGS. 8D, I), and scaffolds deliveringmyoblasts and HGF and FGF2 (FIGS. 8E, J). Defects are outlined withdotted lines. At ten days, defects were unresolved and filled withnecrotic debris in all conditions. At 30 days, the laceration injuriesbegan to resolve in all conditions, but myoblasts delivered on scaffoldsin combination with growth factors led to virtually complete resolutionof the defect at this time point. Size bars are shown on thephotomicrographs.

FIGS. 9A-B are bar graphs showing quantitative analysis of remainingdefect area 10 days post injury (FIG. 9A), and 30 days post injury (FIG.9B). Conditions included an injection of myoblasts directly into themuscle (cells[injected]), blank scaffolds, scaffolds releasing growthfactors without cells (HGF/FGF), cells transplanted in scaffolds notreleasing growth factors (cells in scaffold), and scaffolds deliveringmyoblasts and HGF and FGF2 (HGF/FGF2-cells in scaffold). No significantresolution of the defects occurred in any condition at 10 days. Incontrast, at 30 days post injury the defects in muscles treated withscaffolds delivering cells and growth factors were significantly smallerthan in any other condition (* indicates p<0.05, as compared to allother conditions). A less pronounced, but still significant reduction indefect size was also seen in muscles treated with injected cells orscaffolds delivering HGF and FGF2 (^(#) indicates p<0.01 compared toblank scaffolds or cells transplanted on scaffolds not releasing growthfactors). Values represent mean and standard deviation (n=6).

FIGS. 10A-B are photomicrographs showing that the width of regeneratingfibers and number of centrally located nuclei at 30 days weresignificantly greater in muscles treated with scaffolds delivering cellsand growth factors (FIG. 10B), as compared to scaffolds delivering onlygrowth factors (FIG. 10A) or any of the other conditions. FIG. 10C is abar graph drawing quantification of fiber width, and FIG. 10D is a bargraph showing the number of centrally located nuclei per fiber length.Fiber width was increased with myoblast injection or treatment withscaffolds releasing HGF and FGF2 (^(#) indicates p<0.01 to blankscaffolds or scaffolds transplanting cells without growth factors), andwas most dramatically increased by treatment with scaffolds deliveringmyoblasts and growth factors (* indicates p<0.001 compared to all otherconditions). Increased centrally located nuclei per muscle length wasobserved only when scaffolds containing myoblasts and HGF/FGF2 were usedto treat muscle injury. Values represent mean and standard deviation(n=6).

FIGS. 11 A-D are photomicrographs of muscle tissue. Photomicrographs atlow power (FIGS. 11A-B), and high power (FIGS. 11C-D) of tissue sectionswere immunostained to identify donor myoblasts (positive staining forβ-galactocidase) in the regenerating tissues. Injection of cells (FIGS.11B, D) led to minimal donor cell incorporation into host musculature.In contrast, transplantation of cells on scaffolds releasing growthfactors leads to extensive incorporation of donor cells into theregenerating muscle tissue (FIGS. 11A, C). Size bars are shown on thephotomicrographs.

FIG. 12 is a bar graph showing endothelial cell migration out ofalginate gel scaffolds that contain VEGF compared to scaffolds withoutVEGF.

FIGS. 13A-B are photographs of mouse hindlimbs showing blood perfusionbefore and after surgery. Transplanting cells within the gel matrixenhanced the recovery of blood flow (FIG. 13A) compared to delivery ofcells via intramuscular injection (FIG. 13B). FIG. 13C is a line graphshowing that transplanting EPC and OEC within the gel matrix led to thecomplete recovery of blood flow in the hind limb in which the artery wasligated. FIG. 13D is a bar graph showing recovery of blood perfusion.Hindlimbs subjected to surgery were visually examined, and grouped asnormal (displaying no discrepancy in color or limb integrity as comparedto non-ischemic hindlimbs of the same animal), or presenting onenecrotic toe, multiple necrotic toes, or a complete necrotic foot.

FIG. 14A is a scatter plot and FIGS. 14B-C are line graphs showing thatGM-CSF delivery from PLG scaffolds enhances the in vivo recruitment andexpansion of DCs. FIG. 14A shows FACS plots of cells positive for the DCmarkers, CD86 and CD11c, after isolation from GM-CSF loaded scaffoldsand blank scaffolds. FIG. 14B shows the percentage of DCs isolated fromGM-CSF loaded scaffolds (-▪-) and blank scaffolds(-□-). FIG. 14C showsthe total number of DCs isolated from GM-CSF loaded scaffolds (-▪-) andblank scaffolds(-□-). GM-CSF scaffolds were loaded with 3 μg of therecombinant protein.

FIG. 15A-B are bar graphs showing that DC infiltration is enhanced withan increase in the GM-CSF dose incorporated into PLG scaffolds. FIG. 15Ashows the percentage of CD11c+CD86+ dendritic cells isolated from PLGscaffolds in response to delivery of 1, 3 and 7 μg of GM-CSF (n=4), andFIG. 15B shows the cellular density of CD11c+CD86+ dendritic cellsnormalized by the control. Scaffolds were explanted from subcutaneouspockets at Day 14.

FIG. 16A is a schematic diagram of an in vivo DC tracking assay. DCs arerecruited to FITC painted PLG matrices, implanted subcutaneously intothe backs of C57B6 mice, where they pick up FITC molecules and emigrateto the lymph nodes (LN) as FITC+DCs. Local GM-CSF delivery fromfluoroscein (FITC) painted PLG matrices allows for the sustainedtransport of matrix derived DCs to the draining lymph nodes for extendedperiods. FIG. 16B is a series of scatter plots showing representativeFACS data of CD 11c+FITC+DCs in the inguinal lymph nodes at 2,7 and 14days after the implantation of Blank and GM-CSF loaded PLG matrices.FIG. 16C is a line graph showing the total number of FITC+DCs in theinguinal lymph nodes of C57B6 mice at days 2, 4, 7, 14 and 28 days afterthe implantation of blank and GM-CSF loaded scaffolds.

DETAILED DESCRIPTION OF THE INVENTION

Regenerative medical technologies are devices and methods that repair orreplace diseased or defective tissues or organs. Tissue engineering isthe application of the principles and methods of engineering and thelife sciences to the development of biological substitutes to restore,maintain or improve function of bodily structures and tissues, or toselectively promote the destruction of undesired tissues. It involvesthe development of methods to build biological substitutes assupplements or alternatives to whole organ or tissue transplantation, orthe development of strategies to manipulate tissues in vivo. The use ofliving cells and/or extracellular matrix (ECM) components in thedevelopment of implantable parts or devices is an attractive approach torestore or to replace function. The methods and devices are useful togenerate functional biological structure de novo or to regenerate organsin situ, as well as to restore or supplement tissue function. Thedevices are placed into or adjacent to a particular diseased or injuredtissue in the body or broadly dispersed throughout a tissue in the body.The device makes direct contact with the tissue to be treated orcontains cells that migrate to nearby or remote tissue targets followingresidence in the device.

Cell Populations

Scaffold structures are seeded with one or more populations of purifiedor isolated cells. The term “isolated” used in reference to a cell type,e.g., a stem cell means that the cell is substantially free of othercell types or cellular material with which it naturally occurs. Forexample, a sample of cells of a particular tissue type or phenotype is“substantially pure” when it is at least 60% of the cell population.Preferably, the preparation is at least 75%, more preferably at least90%, and most preferably at least 99% or 100%, of the cell population.Purity is measured by any appropriate standard method, for example, byfluorescence-activated cell sorting (FACS). Optionally, the device isseeded with two or more substantially pure populations of cells. Thepopulations are spatially or physically separated, e.g., one populationis encapsulated, or the cells are allowed to come into with one another.The scaffold or structural support not only provides a surface uponwhich cells are seeded/attached but indirectly affectsproduction/education of cell populations by housing a second (third, orseveral) cell population(s) with which a first population of cellsassociates (cell-cell adhesion). Such “accessory” cell populationssecrete desirable cytokines, growth factors or other signalingmolecules, and/or deposit appropriate extracellular matrix proteins.Cytokines are small secreted proteins which mediate and regulateimmunity, inflammation, and hematopoiesis. Cytokines can act over shortdistances and short time spans and at very low concentration. They actby binding to specific membrane receptors, which then signal cells viasecond messengers, often tyrosine kinases, to alter its behavior (e.g.,cell function and/or gene expression). Responses to cytokines includeincreasing or decreasing expression of membrane proteins (includingcytokine receptors), proliferation, and secretion of effector molecules.Such molecules are also referred to as lymphokines (cytokines made bylymphocytes), monokines (cytokines made by monocytes), chemokines(cytokines with chemotactic activities), and interleukins (cytokinesmade by one leukocyte and acting on other leukocytes). Cytokines act onthe cells that secrete them (autocrine action), on nearby cells(paracrine action), or on distant cells (endocrine action).

A stem cell is an undifferentiated cell that differentiates into amature functional tissue specific cell upon contact with appropriatemicroenvironment, e.g., growth factors and other differentiating agents.The devices/scaffold described herein represent such a microenvironment.Each device constitutes a factory that attracts/accepts, reproduces,sustains, educates, and sends forth to surrounding bodily tissuestissue-specific cells that are capable of colonizing and regeneratingdamaged tissue. In addition to stem cells, the scaffolds houseprogenitor cells, differentiated cells, support cells, modified cells(e.g., genetically modified (e.g., by DNA delivery (by plasmid orvirus), or by siRNA, μRNA) to produce exogenous proteins or chemicallymodified with drugs to enhance or suppress specific signaling pathways(e.g., protein kinases) or genetic regulatory pathways (e.g., upregulateor downregulate activity of master transcription factors) that areinvolved in a variety of cell fate decisions, or surface modified withligands or growth factors and morphogens, or small molecule mimics ofthe same, to promote specific adhesive interactions with other cells,including immune cells, or provide autocrine or paracrine signaling,fused cell populations, fibroblasts, chondrocytes, osteoblasts,myoblasts, endothelial, smooth muscle and neuronal cells.

Differentiated cells are reprogrammed to an embryonic-like state bytransfer of nuclear contents into oocytes or by fusion with embryonicstem (ES) cells. For example, the induction of pluripotent stem cellsfrom mouse embryonic or adult fibroblasts is accomplished by introducingone or more of the following factors, Oct3/4, Sox2, c-Myc, Klf4, andNanog. Following contact with such factors, differentiated cells arereprogrammed and exhibit the morphology and growth properties of stemcells, e.g., ES cells, and express stem cell marker genes.

The scaffolds are seeded in vitro or in vivo. For example, scaffolds areseeded by incubating the structure in a solution containing the cells.Alternatively, cells are injected/titrated into the scaffold orrecruited to migrate into the device. In yet another example, thescaffold is built in stages with each layer of the multicomponentscaffold being seeded prior to laying down of another layer or beforeadherences of another pre-formed component. Different cell types, e.g.,stem vs. differentiated, support vs. therapeutic, are optionallyco-resident in the scaffold housing. Cells optionally vary in phenotype,e.g., differentiation state, activation state, metabolic state, orfunctional state. The scaffolds are suitable for use with any cell typethat one may want to transplant. Such cells include but are not limitedto, various stem cell populations (embryonic stem cells differentiatedinto various cell types), bone marrow or adipose tissue derived adultstem cells, mesenchymal stem cells, cardiac stem cells, pancreatic stemcells, endothelaila progenitor cells, outgrowth endothelial cells,dendritic cells, hematopoietic stem cells, neural stem cells, satellitecells, side population cells. Such cells may further include but are notlimited to, differentiated cell populations including osteoprogenitorsand osteoblasts, chondrocytes, keratinocytes for skin, tenocytes fortendon, and intestinal epithelial cells, smooth muscle cells, cardiacmuscle cells, epithelial cells, endothelial cells, urothelial cells,fibroblasts, myoblasts, chondroblasts, osteoclasts, hepatocytes, bileduct cells, pancreatic islet cells, thyroid, parathyroid, adrenal,hypothalamic, pituitary, ovarian, testicular, salivary gland cells,adipocytes, and precursor cells. For example, smooth muscle cells andendothelial cells may be employed for muscular, tubular scaffolds, e.g.,scaffolds intended as vascular, esophageal, intestinal, rectal, orureteral scaffolds; chondrocytes may be employed in cartilaginousscaffolds; cardiac muscle cells may be employed in heart scaffolds;hepatocytes and bile duct cells may be employed in liver scaffolds;epithelial, endothelial, fibroblast, and nerve cells may be employed inscaffolds intended to function as replacements or enhancements for anyof the wide variety of tissue types that contain these cells. In generalscaffolds of the invention may comprise any cell population competent toparticipate in regeneration, replacement or repair of a target tissue ororgan. For example, cells are myoblasts for use in muscle regeneration.

Cells are optionally genetically manipulated by the introduction ofexogenous genetic sequences or the inactivation or modification ofendogenous sequences. For example, recombinant genes are introduced tocause the cells to make proteins that are otherwise lacking in the hostor target tissue. Production of scarce but desirable proteins (in thecontext of certain tissues) is augmented by transplanting geneticallyengineered cells. Cells used to seed the scaffold are capable ofdegrading the scaffold matrix over a desired period time in order tomigrate through and out of the scaffold matrix. Scaffold matrices areselected such that they are susceptible to degradation by certain celltypes seeded within the matrix. For example, scaffold materials andcells are selected and designed such that all or some of the cellsseeded within the scaffolds require a certain desired period of timedegrade the scaffold sufficiently to migrate through it and reach thesurrounding tissue. The delay in the release of the cells to thesurrounding tissue is controlled by varying the composition of thescaffold, to allow optimal time to signal the cells to multiply,differentiate, or achieve various phenotypes. General mammalian cellculture techniques, cell lines, and cell culture systems are describedin Doyle, A., Griffiths, J. B., Newell, D. G., (eds.) Cell and TissueCulture: Laboratory Procedures, Wiley, 1998, the contents of which areincorporated herein by reference.

Cells secrete enzymes that degrade the material of the scaffold, therebycontrolling the rate at which cells exit the scaffold. For example,migrating cells typically secrete collagenases and plasmin to degradetheir matrix and allow cell movement. The rate of cells exiting may thusbe regulated by controlling the density and susceptibility to theseenzymes of oligopeptides used as either cross-links in the material oras components of the main chains. Certain materials are degraded in apreprogrammed manner independent of cell action (e.g. hydrolyticdegradation of poly(lactide-co glyolide) as a degradable scaffold. Thescaffolds may be prepared such that the degradation time may becontrolled by using a mixture of degradable components in proportions toachieve a desired degradation rate. Alternatively, the cells themselvesaid in the degradation. For example, scaffold compositions are sensitiveto degradation by materials secreted by the cells themselves that areseeded within the scaffold. One example of this is the use ofmetalloproteinase (MMP)-sensitive substrate in the scaffold matrix;cells exit when the seeded cells have secreted sufficient MMP to begindegradation of the matrix.

Cells incubated in the scaffold are educated and induced to migrate outof the scaffold to directly affect a target tissue, e.g., and injuredtissue site. For example, stromal vascular cells and smooth muscle cellsare useful in sheetlike structures are used for repair of vessel-likestructures such as blood vessels or layers of the body cavity. Suchstructures are used to repair abdominal wall injuries or defects such asgastroschisis. Similarly, sheetlike scaffolds seeded with dermal stemcells and/or keratinocytes are used in bandages or wound dressings forregeneration of dermal tissue.

Scaffold Compositions and Architecture

Components of the scaffolds are organized in a variety of geometricshapes (e.g., beads, pellets), niches, planar layers (e.g., thinsheets). For example, multicomponent scaffolds are constructed inconcentric layers each of which is characterized by different physicalqualities (% polymer, % crosslinking of polymer, chemical composition ofscaffold, pore size, porosity, and pore architecture, stiffness,toughness, ductility, viscoelasticity, and or composition of bioactivesubstances such as growth factors, homing/migration factors,differentiation factors. Each niche has a specific effect on a cellpopulation, e.g., promoting or inhibiting a specific cellular function,proliferation, differentiation, elaboration of secreted factors orenzymes, or migration. Cells incubated in the scaffold are educated andinduced to migrate out of the scaffold to directly affect a targettissue, e.g., and injured tissue site. For example, stromal vascularcells and smooth muscle cells are useful in sheetlike structures areused for repair of vessel-like structures such as blood vessels orlayers of the body cavity. For example, such structures are used torepair abdominal wall injuries or defects such as gastroschisis.Similarly, sheetlike scaffolds seeded with dermal stem cells and/orkeratinocytes are used in bandages or wound dressings for regenerationof dermal tissue. The device is placed or transplanted on or next to atarget tissue, in a protected location in the body, next to bloodvessels, or outside the body as in the case of an external wounddressing. Devices are introduced into or onto a bodily tissue using avariety of known methods and tools, e.g., spoon, tweezers or graspers,hypodermic needle, endoscopic manipulator, endo- or trans-vascular-catheter, stereotaxic needle, snake device, organ-surface-crawling robot(U.S. Patent Application 20050154376; Ota et al., 2006, Innovations1:227-231), minimally invasive surgical devices, surgical implantationtools, and transdermal patches. Devices can also be assembled in place,for example by senquentially injecting or inserting matrix materials.Scaffold devices are optionally recharged with cells or with bioactivecompounds, e.g., by sequential injection or spraying of substances suchas growth factors or differentiation factors.

A scaffold or scaffold device is the physical structure upon which orinto which cells associate or attach, and a scaffold composition is thematerial from which the structure is made. For example, scaffoldcompositions include biodegradable or permanent materials such as thoselisted below. The mechanical characteristics of the scaffold varyaccording to the application or tissue type for which regeneration issought. It is biodegradable (e.g., collagen, alginates, polysaccharides,polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA),or poly(lactide-co-glycolide) (PLGA) or permanent (e.g., silk). In thecase of biodegradable structures, the composition is degraded byphysical or chemical action, e.g., level of hydration, heat or ionexchange or by cellular action, e.g., elaboration of enzyme, peptides,or other compounds by nearby or resident cells. The consistency variesfrom a soft/pliable (e.g., a gel) to glassy, rubbery, brittle, tough,elastic, stiff. The structures contain pores, which are nanoporous,microporous, or macroporous, and the pattern of the pores is optionallyhomogeneous, heterogenous, aligned, repeating, or random.

Alginates are versatile polysaccharide based polymers that may beformulated for specific applications by controlling the molecularweight, rate of degradation and method of scaffold formation. Couplingreactions can be used to covalently attach bioactive epitopes, such asthe cell adhesion sequence RGD to the polymer backbone. Alginatepolymers are formed into a variety of scaffold types. Injectablehydrogels can be formed from low MW alginate solutions upon addition ofa cross-linking agents, such as calcium ions, while macroporousscaffolds are formed by lyophilization of high MW alginate discs.Differences in scaffold formulation control the kinetics of scaffolddegradation. Release rates of morphogens or other bioactive substancesfrom alginate scaffolds is controlled by scaffold formulation to presentmorphogens in a spatially and temporally controlled manner. Thiscontrolled release not only eliminates systemic side effects and theneed for multiple injections, but can be used to create amicroenvironment that activates host cells at the implant site andtransplanted cells seeded onto a scaffold.

The scaffold comprises a biocompatible polymer matrix that is optionallybiodegradable in whole or in part. A hydrogel is one example of asuitable polymer matrix material. Examples of materials which can formhydrogels include polylactic acid, polyglycolic acid, PLGA polymers,alginates and alginate derivatives, gelatin, collagen, agarose, naturaland synthetic polysaccharides, polyamino acids such as polypeptidesparticularly poly(lysine), polyesters such as polyhydroxybutyrate andpoly-epsilon.-caprolactone, polyanhydrides; polyphosphazines, poly(vinylalcohols), poly(alkylene oxides) particularly poly(ethylene oxides),poly(allylamines)(PAM), poly(acrylates), modified styrene polymers suchas poly(4-aminomethylstyrene), pluronic polyols, polyoxamers,poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above,including graft copolymers.

The scaffolds are fabricated from a variety of synthetic polymers andnaturally-occurring polymers such as, but not limited to, collagen,fibrin, hyaluronic acid, agarose, and laminin-rich gels. One preferredmaterial for the hydrogel is alginate or modified alginate material.Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (Munits) and αL-guluronic acid (G units) monomers, which can vary inproportion and sequential distribution along the polymer chain. Alginatepolysaccharides are polyelectrolyte systems which have a strong affinityfor divalent cations (e.g. Ca⁺², Mg⁺², Ba⁺²) and form stable hydrogelswhen exposed to these molecules. See Martinsen A., et al., Biotech. &Bioeng., 33 (1989) 79-89.) For example, calcium cross-linked alginatehydrogels are useful for dental applications, wound dressingschondrocyte transplantation and as a matrix for other cell types.

An exemplary device utilizes an alginate or other polysaccharide of arelatively low molecular weight, preferably of size which, afterdissolution, is at the renal threshold for clearance by humans, e.g.,the alginate or polysaccharide is reduced to a molecular weight of 1000to 80,000 daltons. Preferably, the molecular mass is 1000 to 60,000daltons, particularly preferably 1000 to 50,000 daltons. It is alsouseful to use an alginate material of high guluronate content since theguluronate units, as opposed to the mannuronate units, provide sites forionic crosslinking through divalent cations to gel the polymer. U.S.Pat. No. 6,642,363, incorporated herein by reference discloses methodsfor making and using polymers containing polysachharides such asalginates or modified alginates that are particularly useful for celltransplantation and tissue engineering applications.

Useful polysaccharides other than alginates include agarose andmicrobial polysaccharides such as those listed in the table below.

Polysaccharide Scaffold Compositions Polymers^(a) Structure FungalPullulan (N) 1,4-; 1,6-α-D-Glucan Scleroglucan (N) 1,3; 1,6-α-D-GlucanChitin (N) 1,4-β-D-Acetyl Glucosamine Chitosan (C)1,4-β.-D-N-Glucosamine Elsinan (N) 1,4-; 1,3-α-D-Glucan BacterialXanthan gum (A) 1,4-β.-D-Glucan with D-mannose; D-glucuronic Acid asside groups Curdlan (N) 1,3-β.-D-Glucan (with branching) Dextran (N)1,6-α-D-Glucan with some 1,2; 1,3-; 1,4-α- linkages Gellan (A)1,4-β.-D-Glucan with rhamose, D-glucuronic acid Levan (N)2,6-β-D-Fructan with some β-2,1-branching Emulsan (A)Lipoheteropolysaccharide Cellulose (N) 1,4-β-D-Glucan ^(a)N—neutral, A =anionic and C = cationic.

The scaffolds of the invention are porous or non-porous. For example,the scaffolds are nanoporous having a diameter of less than about 10 nm;microporous wherein the diameter of the pores are preferably in therange of about 100 nm-20 μm; or macroporous wherein the diameter of thepores are greater than about 20 μm, more preferably greater than about100 μm and even more preferably greater than about 400μm. In oneexample, the scaffold is macroporous with aligned pores of about400-500μm in diameter. The preparation of polymer matrices having thedesired pore sizes and pore alignments are described in the Examples.Other methods of preparing porous hydrogel products are known in theart. (U.S. Pat. No. 6,511,650 incorporated herein by reference).

Bioactive Compositions

The device includes one or more bioactive compositions. Bioactivecompositions are purified naturally-occurring, synthetically produced,or recombinant compounds, e.g., polypeptides, nucleic acids, smallmolecules, or other agents. The compositions described herein arepurified. Purified compounds are at least 60% by weight (dry weight) thecompound of interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight thecompound of interest. Purity is measured by any appropriate standardmethod, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

The bioactive composition alters a function, e.g., level ofdifferentiation, state of activation, motility, or gene expression, of acell. For example, at least one cell adhesion molecule is incorporatedinto or onto the polymer matrix. Such molecules are incorporated intothe polymer matrix prior to polymerization of the matrix or afterpolymerization of the matrix. Examples of cell adhesion moleculesinclude but are not limited to peptides, proteins and polysaccharides.More specifically, cell adhesion molecules include fibronectin, laminin,collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan,agrin, bone sialoprotein, cartilage matrix protein, fibronogen, fibrin,fibulin, mucins, entactin, osteopontin, plasminogen, restrictin,serglycin, SPARC/osteonectin, versican, von Willebrand Factor,polysaccharide heparin sulfate, connexins, collagen, RGD (Arg-Gly-Asp)and YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides and cyclic peptides,glycosaminoglycans (GAGs), hyaluronic acid (HA), condroitin-6-sulfate,integrin ligands, selectins, cadherins and members of the immunoglobulinsuperfamily. Other examples include neural cell adhesion molecules(NCAMs), intercellular adhesion molecules (ICAMs), vascular celladhesion molecule (VCAM-1), platelet-endothelial cell adhesion molecule(PECAM-1), L1, and CHL1.

Examples of some of these molecules and their function are shown in thefollowing table.

ECM Proteins and peptides and role in cell function Protein SequenceSeq. ID No: Role Fibronectin RGDS Adhesion LDV Adhesion REDV AdhesionVitronectin RGDV Adhesion Laminin A LRGDN SEQ ID NO: 7 Adhesion IKVAVSEQ ID NO: 8 Neurite extension Laminin B1 YIGSR SEQ ID NO: 9 Adhesion ofmany cells, via 67 kD laminin receptor PDSGR SEQ ID NO: 10 AdhesionLaminin B2 RNIAEIIKDA SEQ ID NO: 11 Neurite extension Collagen 1 RGDTAdhesion of most cells DGEA Adhesion of platelets, other cells Thrombo-RGD Adhesion of spondin most cells VTXG Adhesion of plateletsHubbell, J A (1995): Biomaterials in tissue engineering.

Bio/Technology 13:565-576. One-letter abbreviations of amino acids areused, X stands for any amino acid.

Additional examples of suitable cell adhesion molecules are shown below.

Amino acid sequences specific for proteoglycan binding fromextracellular matrix proteins SEQ. SEQUENCE ID. NO. PROTEIN XBBXBX*Consensus sequence PRRARV Fibronectin YEKPGSPPREVVPRPRPGV FibronectinRPSLAKKQRFRHRNRKGYRSQRGHSRGR Vitronectin rIQNLLKITNLRIKFVK Laminin

Particularly preferred cell adhesion molecules are peptides or cyclicpeptides containing the amino acid sequence arginine-glycine-asparticacid (RGD) which is known as a cell attachment ligand and found invarious natural extracellular matrix molecules. A polymer matrix withsuch a modification provides cell adhesion properties to the scaffold,and sustains long-term survival of mammalian cell systems, as well assupporting cell growth and differentiation.

Coupling of the cell adhesion molecules to the polymer matrix isaccomplished using synthetic methods which are in general known to oneof ordinary skill in the art and are described in the examples.Approaches to coupling of peptides to polymers are discussed in Hiranoand Mooney, Advanced Materials, p. 17-25 (2004). Other useful bondingchemistries include those discussed in Hermanson, BioconjugateTechniques, p. 152-185 (1996), particularly by use of carbodiimidecouplers, DCC and DIC (Woodward's Reagent K). Since many of the celladhesion molecules are peptides, they contain a terminal amine group forsuch bonding. The amide bond formation is preferably catalyzed by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is a watersoluble enzyme commonly used in peptide synthesis. The density of celladhesion ligands, a critical regulator of cellular phenotype followingadhesion to a biomaterial. (Massia and Hubbell, J. Cell Biol.114:1089-1100, 1991; Mooney et al., J. Cell Phys. 151:497-505, 1992; andHansen et al., Mol. Biol. Cell 5:967-975, 1994) can be readily variedover a 5-order of magnitude density range.

Device Construction

The scaffold structure is constructed out of a number of differentrigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline,or fluid compositions such as peptide polymers, polysaccharides,synthetic polymers, hydrogel materials, ceramics (e.g., calciumphosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans,metals and metal alloys. The compositions are assembled into cellscaffold structures using methods known in the art, e.g., injectionmolding, lyophillization of preformed structures, printing,self-assembly, phase inversion, solvent casting, melt processing, gasfoaming, fiber forming/processing, particulate leaching or a combinationthereof. The assembled devices are then implanted or administered to thebody of an individual to be treated.

The device is assembled in vivo in several ways. The scaffold is madefrom a gelling material, which is introduced into the body in itsungelled form where it gells in situ. Exemplary methods of deliveringdevice components to a site at which assembly occurs include injectionthrough a needle or other extrusion tool, spraying, painting, or methodsof deposit at a tissue site, e.g., delivery using an application deviceinserted through a cannula. In one example, the ungelled or unformedscaffold material is mixed with bioactive substances and cells prior tointroduction into the body or while it is introduced. The resultant invivo/in situ assembled scaffold contains a mixture of these substancesand cells.

In situ assembly of the scaffold occurs as a result of spontaneousassociation of polymers or from synergistically or chemically catalyzedpolymerization. Synergistic or chemical catalysis is initiated by anumber of endogenous factors or conditions at or near the assembly site,e.g., body temperature, ions or pH in the body, or by exogenous factorsor conditions supplied by the operator to the assembly site, e.g.,photons, heat, electrical, sound, or other radiation directed at theungelled material after it has been introduced. The energy is directedat the scaffold material by a radiation beam or through a heat or lightconductor, such as a wire or fiber optic cable or an ultrasonictransducer. Alternatively, a shear-thinning material, such as anampliphile, is used which re-cross links after the shear force exertedupon it, for example by its passage through a needle, has been relieved.

Suitable hydrogels for both in vivo and ex vivo assembly of scaffolddevices are well known in the art and described, e.g., in Lee et al.,2001, Chem. Rev. 7:1869-1879. The peptide amphiphile approach toself-assembly assembly is described, e.g., in Hartgerink et al., 2002,Proc. Natl. Acad. Sci. U. S. A. 99:5133-5138. A method for reversiblegellation following shear thinning is exemplied in Lee et al., 2003,Adv. Mat. 15:1828-1832

A multiple compartment device is assembled in vivo by applyingsequential layers of similarly or differentially doped gel or otherscaffold material to the target site. For example, the device is formedby sequentially injecting the next, inner layer into the center of thepreviously injected material using a needle, forming concentricspheroids. Non-concentric compartments are formed by injecting materialinto different locations in a previously injected layer. A multi-headedinjection device extrudes compartments in parallel and simultaneously.The layers are made of similar or different scaffolding compositionsdifferentially doped with bioactive substances and different cell types.Alternatively, compartments self-organize based on theirhydro-philic/phobic characteristics or on secondary interactions withineach compartment.

Compartmentalized Device

In certain situations, a device containing compartments with distinctchemical and/or physical properties is useful. Such a configuration isparticularly useful in maintaining for long time periods the “stemness”of a population of cells, while simultaneously pushing daughter cells tomultiply rapidly and differentiate appropriately for participation intissue regeneration. This system provides a long-term (e.g., months toyears) stream of cells from the device. For example, an innercompartment maintains a quiescent population of multipotent stem cells,and a second compartment promotes a high rate of proliferation of thecells while inhibiting differentiation. The cells that migrate out ofthe second device to the surrounding tissue are instructed, as they passthrough a third compartment, to differentiate appropriately. The slowlycycling cells in the inner population repopulate the intermediatecompartment with a portion of their daughters, where the transient,amplifying cells provide the bulk of regenerative cells.

A comparmentalized device is designed and fabricated using differentcompositions or concentrations of compositions for each compartment. Forexample, the stem cell population is encapsulated within hydrogels,using standard encapsulation techniques (e.g., alginate microbeadformation). This first hydrogel contains factors required to maintainthe multipotent nature of the stem cells, either by their covalentcoupling to the polymer forming the gel or by their slow and sustainedrelease from the gel. This compartment is then coated with a secondlayer of gel (e.g., double layered alginate microbeads) that containsfactors that do not maintain stemness, but instead promote the stemcells to rapidly proliferate and generate large numbers of morespecialized daughter cells. This second compartment is formed from thesame material that contains distinct factors (e.g., morphogens, growthfactors, adhesion ligands), the same material in a distinct form (e.g.,varying mechanical properties or porosity), or a completely differentmaterial that provides appropriate chemical/physical properties.

Alternatively, the compartments are fabricated individually, and thenadhered to each other (e.g., a “sandwich” with an inner compartmentsurrounded on one or all sides with the second compartment). This latterconstruction approach is accomplished using the intrinsic adhesivenessof each layer for the other, diffusion and interpenetration of polymerchains in each layer, polymerization or cross-linking of the secondlayer to the first, use of an adhesive (e.g., fibrin glue), or physicalentrapment of one compartment in the other. The compartmentsself-assemble and interface appropriately, either in vitro or in vivo,depending on the presence of appropriate precursors (e.g., temperaturesensitive oligopeptides, ionic strength sensitive oligopeptides, blockpolymers, cross-linkers and polymer chains (or combinations thereof),and precursors containing cell adhesion molecules that allowcell-controlled assembly). Multiple compartments are designed to stagethe proliferation and specialization of the desired cells appropriately.In addition, the device is designed to have a number of compartments, inwhich cells enter in parallel, in contrast to serially passing throughall compartments. The different compartments each induce distinct fatesfor the contained cells, and in this manner provide multiple specializeddaughter cell populations from a single, starting stem cell population.An individual with ordinary skill in the art of stem cell biology andbiomaterials can readily derive a number of potentially useful designsfor a given starting cell type and desired daughter cell output.

Alternatively, the compartmentalized device is formed using a printingtechnology. Successive layers of a scaffold precursor doped withbioactive substances and/or cells is placed on a substrate then crosslinked, for example by self-assembling chemistries. When the crosslinking is controlled by chemical-, photo- or heat-catalyzedpolymerization, the thickness and pattern of each layer is controlled bya masque, allowing complex three dimensional patterns to be built upwhen un-cross-linked precursor material is washed away after eachcatalyzation. (W T Brinkman et al., Photo-cross-linking of type 1collagen gels in the presence of smooth muscle cells: mechanicalproperties, cell viability, and function. Biomacromolecules, 2003July-August; 4(4): 890-895.; W. Ryu et al., The construction ofthree-dimensional micro-fluidic scaffolds of biodegradable polymers bysolvent vapor based bonding of micro-molded layers. Biomaterials, 2007February; 28(6): 1174-1184; Wright, Paul K. (2001). 21st Centurymanufacturing. New Jersey: Prentice-Hall Inc. ) Complex,multi-compartment layers are also built up using an inkjet device which“paints” different doped-scaffold precursors on different areas of thesubstrate. Julie Phillippi (Carnegie Mellon University) presentation atthe annual meeting of the American Society for Cell Biology on Dec. 10,2006; Print me a heart and a set of arteries, Aldhouse P., New Scientist13 Apr. 2006 Issue 2547 p 19.; Replacement organs, hot off the press, C.Choi, New Scientist, 25 Jan. 2003, v2379. These layers are built-up intocomplex, three dimensional compartments. The device is also built usingany of the following methods: Jetted Photopolymer, Selective LaserSintering, Laminated Object Manufacturing, Fused Deposition Modeling,Single Jet Inkjet, Three Dimensional Printing, or Laminated ObjectManufacturing.

Growth Factors and Incorporation of Compositions into/onto a ScaffoldDevice

Bioactive substances that influence growth, development, movement, andother cellular functions are introduced into or onto the scaffoldstructures. Such substances include BMP, bone morphogenetic protein;ECM, extracellular matrix proteins or fragments thereof; EGF, epidermalgrowth factor; FGF-2, fibroblast growth factor 2; NGF, nerve growthfactor; PDGF, platelet-derived growth factor; PIGF, placental growthfactor; TGF, transforming growth factor, and VEGF, vascular endothelialgrowth factor. Cell-cell adhesion molecules (cadherins, integrins,ALCAM, NCAM, proteases) are optionally added to the scaffoldcomposition.

Exemplary growth factors and ligands are provided in the tables below.

Growth factors used for angiogenesis Growth factor Abbreviation Relevantactivities Vascular endothelial VEGF Migration, proliferation and growthfactor survival of ECs Basic fibroblast bFGF-2 Migration, proliferationand survival growth factor of ECs and many other cell typesPlatelet-derived PDGF Promotes the maturation of blood growth factorvessels by the recruitment of smooth muscle cells Angiopoietin-1 Ang-1Strengthens EC-smooth muscle cell interaction Angiopoietin-2 Ang-2Weakens EC-smooth muscle cell interaction Placental growth PIGFStimulates angiogenesis factor Transforming TGF Stabilizes new bloodvessels by growth factor promoting matrix deposition

Growth factors used for bone regeneration Growth factor AbbreviationRelevant activities Transforming growth TGF-β Proliferation anddifferentiation factor-β of bone-forming cells Bone morphogenetic BMPDifferentiation of bone-forming protein cells Insulin-like growth IGF-1Stimulates proliferation of factor osteoblasts and the synthesis of bonematrix Fibroblast growth FGF-2 Proliferation of osteoblasts factor-2Platelet-derived PDGF Proliferation of osteoblasts growth factor

Growth factors used for wound healing Growth Factor AbbreviationRelevant activities Platelet-derived PDGF Active in all stages ofhealing growth factor process Epidermal growth EGF Mitogenic forkeratinocytes factor Transforming TGF-β Promotes keratinocyte migration,growth factor-β ECM synthesis and remodeling, and differentiation ofepithelial cells Fibroblast FGF General stimulant for wound healinggrowth factor

Growth Factors Used for Tissue- Engineering Moleular Representativesupplier Growth factor Abbreviation weight (kDa) Relevant activities ofrH growth factor Epidermal growth EGF 6.2 Proliferation of epithelial,mesenchymal, and PeproTech Inc. (Rocky factor fibroblast cells Hill, NJ,USA) Platelet-derived PDGF-AA 28.5 Proliferation and chemoattractantagent for PeproTech Inc. growth factor PDGF-AB 25.5 smooth muscle cells;extracellular matrix PDGF-BB 24.3 synthesis and deposition TransformingTFG-α 5.5 Migration and proliferation of keratinocytes; PeproTech Inc.growth factor-α extracellular matrix synthesis and depositionTransforming TGF-β 25.0 Proliferation and differentiation of bonePeproTech Inc. growth factor-β forming cells; chemoattractant forfibroblasts Bone morphogenetic BMP-2 26.0 Differentiation and migrationof bone Cell Sciences Inc. protein BMP-7 31.5 forming cells (Norwood,MA, USA) Basic fibroblast bFGF/FGF- 17.2 Proliferation of fibroblastsand initiation of PeproTech Inc. growth factor 2 angiogenesis Vascularendothelial VEGF₁₆₅ 38.2 Migration, proliferation, and survival ofPeproTech Inc. growth factor endothelial cells rH. recombinant human

Immobilized ligands used in tissue engineering Immobilized ligand* ECMmolecule source Application RGD Multiple ECM molecules, Enhance bone andcartilage tissue formation in vitro and in including fibronectin, vivovitronectin, laminin, collagen and Regulate neurite outgrowth in vitroand in vivo thrombospondin Promote myoblast adhesion, proliferation anddifferentiation Enhance endothelial cell adhesion and proliferationIKVAV YIGSR Laminin Regulate neurite outgrowth in vitro and in vivoRNIAEIIKDI Recombinant fibronectin Fibronectin Promote formulation offocal contacts in pre-osteoblasts fragment (FNIII₇₋₁₀) Ac-GCRDGPQ-Common MMP substrates, (e.g. Encourage cell-mediated proteolyticdegradation, GIWGQDRCG collagen, fibronectin, laminin) remodeling andbone regeneration (with RGD and BMP-2 presentation) in vivo *Sequencesare given in single-letter amino acid code. MMP, matrixmetalloproteinase.

The release profiles of bioactive substances from scaffold devices iscontrolled by both factor diffusion and polymer degradation, the dose ofthe factor loaded in the system, and the composition of the polymer.Similarly, the range of action (tissue distribution) and duration ofaction, or spatiotemporal gradients of the released factors areregulated by these variables. The diffusion and degradation of thefactors in the tissue of interest is optionally regulated by chemicallymodifying the factors (e.g., PEGylating growth factors). In both cases,the time frame of release determines the time over which effective celldelivery by the device is desired.

Carrier systems for tissue regeneration are described in the tablebelow.

Polymeric carriers used to deliver various growth factors and the typeof tissues regenerated Growth factor Carrier Tissue regenerated EGFGelatin Dermis PET suture Tendon PVA sponge Dermis PDGF Chitosan-PLLAscaffold Craniofacial bone CMC gel Dermis Fibrin Ligament Porous HA LongBone TGF-β Alginate Cartilage PLA Long Bone CaP-titanium meshCraniofacial bone Polyoxamer; PEO gel Dermis rhBMP-2 Collagen spongeLong bone HA-TCP granules Craniofacial bone HA-collagen Spinal bonePLA-DX-PEG Long bone Ectopic and hip bone rHBMP-7 HA Spinal boneCollagen-CMC Spinal bone Porous HA Craniofacial bone bFGF ChitosanDermis Heparin-alginate Blood vessels EVAc microspheres Blood vesselsFibrin matrices Blood vessels VEGF PLG scaffold Blood vessels PLGscaffold Blood vessels PLG microspheres Blood vessels Fibrin mesh Bloodvessels Abbreviations: PET, poly (ethylene terepthalate); PVA, polyvinylalcohol; PLLA, poly(L-lactic acid); CMC, carboxymethylcellulose; HA,hydroxyapatite; PLA, poly(D,L-lactic acid); CaP, calcium phosphate; PEO,poly (ethylene oxide); TCP, tricalcium phosphate; PEG, poly(ethyleneglycol); -DX-, -p-dioxanone-; EVAc, ethylene vinyl acetate; PLG, poly(lactide-co-glycolide).

The bioactive substances are added to the scaffold compositions usingknown methods including surface absorption, physical immobilization,e.g., using a phase change to entrap the substance in the scaffoldmaterial. For example, a growth factor is mixed with the scaffoldcomposition while it is in an aqueous or liquid phase, and after achange in environmental conditions (e.g., pH, temperature, ionconcentration), the liquid gels or solidifies thereby entrapping thebioactive substance. Alternatively, covalent coupling, e.g., usingalkylating or acylating agents, is used to provide a stable, longtermpresentation of a bioactive substance on the scaffold in a definedconformation. Exemplary reagents for covalent coupling of suchsubstances are provided in the table below.

Methods to covalently couple peptides/proteins to polymers FunctionalGroup of Coupling reagents Reacting groups on Polymer and cross-linkerproteins/peptides —OH Cyanogen bromide (CNBr) —NH₂ Cyanuric chloride4-(4,6-Dimethoxy-1,3,5- triazin-2-yl)-4-methyl- morpholinium chloride(DMT-MM) —NH₂ Diisocyanate compounds —NH₂ Diisothoncyanate compounds —OHGlutaraldehyde Succinic anhydride —NH₂ Nitrous Acid —NH₂ Hydrazine +nitrous acid —SH —Ph-OH —NH₂ Carbodiimide compounds —COOH (e.g., EDC,DCC)[a] DMT-MM —COOH Thionyl chloride —NH₂ N-hydroxysuccinimideN-hydroxysulfosuc- cinimide + EDC —SH Disulfide compound —SH [a]EDC:1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; DCC:dicyclohexylcarbodiimide

Bioactive substances are capable of inducing migration of thetransplanted cells and their progeny out of the polymer matrix. Otherpreferred bioactive substances are capable of maintaining cellviability, promoting cell proliferation or preventing premature terminaldifferentiation of the transplanted cells. Such bioactive substances areused alone or in combination to achieve the desired result.

Bioactive substances suitable for use in the present invention include,but are not limited to: growth factors, hormones, neurotransmitters,neurotransmitter or growth factor receptors, interferons, interleukins,chemokines, cytokines, colony stimulating factors, chemotactic factors,MMP-sensitive substrate, extracellular matrix components; such as growthhormone, parathyroid hormone (PTH), bone morphogenetic protein (BMP),transforming growth factor-α (TGF-α), TGF-β1, TGF-β2, fibroblast growthfactor (FGF), granulocyte/macrophage colony stimulating factor (GMCSF),epidermal growth factor (EGF), platelet derived growth factor (PDGF),insulin-like growth factor (IGF), scatter factor/hepatocyte growthfactor (HGF), fibrin, collagen, fibronectin, vitronectin, hyaluronicacid, an RGD-containing peptide or polypeptide, an angiopoietin andvascular endothelial cell growth factor (VEGF). Splice variants of anyof the above mentioned proteins, and small molecule agonists orantagonists thereof that may be used advantageously to alter the localbalance of pro and anti-migration and differentiation signals are alsocontemplated herein.

Examples of cytokines as mentioned above include, but are not limited toIL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18,granulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), interferon-γ (γ-IFN), IFN-α, tumornecrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40 ligand.

Suitable bioactive substances useful in accordance with the inventionalso include but are not limited to DNA molecules, RNA molecules,antisense nucleic acids, ribozymes, plasmids, expression vectors, markerproteins, transcription or elongation factors, cell cycle controlproteins, kinases, phosphatases, DNA repair proteins, oncogenes, tumorsuppressors, angiogenic proteins, anti-angiogenic proteins, cell surfacereceptors, accessory signaling molecules, transport proteins, enzymes,anti-bacterial agents, anti-viral agents, antigens, immunogens,apoptosis-inducing agents, anti-apoptosis agents, and cytotoxins.

For some applications, the scaffolds of the invention includes at leastone cell growth factor that prevents premature terminal differentiationof the transplanted cells in the polymer matrix and induces migration ofthe transplanted cells and their progeny out of the polymer matrix. Cellgrowth factors are incorporated into the polymer matrix prior topolymerization of fabrication or may be coupled to the polymer matrixafter polymerization. The choice of growth factor will depend upon thetype of cells and the influence of a particular growth factor on thosecells such that the cells are directed to bypass their normal tendencyto differentiate, and remain in a proliferative phase until a sufficientnumber of cells is attained to regenerate the targeted tissue and forthe cells to have also migrated from the scaffold.

Scaffolds of the invention optionally comprise at least one non-viralgene therapy vector such that either the transplanted cells or hostcells in the vicinity of the implant would take up and express gene thatlead to local availability of the desired factor for a desirable timeframe. Such non-viral vectors include, but are not limited to, cationiclipids, polymers, targeting proteins, and calcium phosphate.

For regeneration of muscular tissue, the cells seeded in the scaffoldare myoblasts and the preferred combination of growth factors is HGF andFGF2. FGF2 is particularly useful in preventing the prematuredifferentiation of the transplanted cells, while HGF induces migrationof the cells from the scaffold. The incorporation of the two growthfactors significantly increased the viability and migration of theseeded myoblasts as discussed below.

Clinical Applications

The devices and methods are useful for generation or regeneration of anumber of different organs and tissue types such as musculoskeletaltissue. In the latter case, environmental cues work in concert withtranscription factors to activate satellite cells, induce them toproliferate and eventually differentiate into mature muscle fibers.Numerous trophic factors play a role as initiators of satellite cellactivation. Of these candidate trophic factors, both hepatocyte growthfactor (HGF) and members of the fibroblast growth factor (FGF) familyhave been demonstrated to have a physiological role in skeletal muscleregeneration. Both types of factors initiate satellite cell activation,stimulate satellite cells to enter the cell cycle in vivo and are potentmitogens for satellite cells. In addition, the receptor for HGF, c-met,is expressed in both quiescent and activated satellite cells, and FGF-2is present in the basement membrane surrounding developing myotubes.Both HGF and FGF2 are heparin binding proteins which depend on heparinsulfate proteoglycans (HSPG) to facilitate receptor activation. WhileHSPG's are ubiquitous on the surface of the cells of mammals, a specificfamily of HSPG's called Syndecans are involved in FGF2 signaling. Inaddition, Syndecan 3 and 4 are expressed on both quiescent and activatedsatellite cells indicating that HGF and FGF2 play importantphysiological roles in regulating satellite cell activation.

Current approaches therapeutically intervene in the muscle regenerativeprocess have been limited by significant drawbacks. The inventionprovides solutions to these drawbacks of earlier methods. Three of suchapproaches are described below. First, cells in the tissues arestimulated to re-enter the cell cycle and repopulate lost or damagedtissues by the injection of growth factors into the site of interest.The second approach is based on the current interest in gene therapy andtargets the intrinsic cell proliferation and differentiation program ofmuscle forming cells. The third approach is based on the delivery ofexogenous cells, expanded in culture, to repair the defect and restorefunction to the tissue. Current strategies related to the third approachinclude direct injection of cells into the injury site, the utilizationof a carrier to provide an artificial matrix for cell delivery, or acombination of cell, matrix and growth factor delivery to increaseregeneration. Cell transplantation approaches have focused on satellitecells, and are gaining growing interest as a potential treatmentalternative for patients with musculodegenerative diseases such asmuscular dystrophy, and for chronic or congenital cardiomyopathies.However, while animal studies were initially promising, attempts totransplant human satellite cells have been disappointing, becausetransplanted myogenic cells underwent rapid and massive necrosis,resulting in less than 5% of transplanted cells incorporating into thehost myofibers after 48 hours.

A solution to problems, e.g., poor survival and integration of myogeniccells into host musculature, is addressed by the compositions andmethods described herein. The invention provides a new approach totissue engineering and regenerative medicine. The systems mediate andregulate delivery of cells on a material that maintains the viability ofthe cells for extended time periods while simultaneously encouragingoutward migration of the cells to populate surrounding host tissue inneed of regeneration. Appropriate combinations of scaffold architecture,adhesion ligands that maintain viability and allow migration, and growthfactors that regulate cell phenotype are used to inform cell behaviorand exert complex control over the fate of the transplanted cells.

In addition to generation or regeneration of muscle tissue, stem cellshave been identified for many different types of tissues, including thehuman heart (Proceedings of the National Academy of Sciences (DOI:10.1073/pnas.0600635103)), delivering them in a way which istherapeutically effective has proven to be a challenge. Neitherintravascular delivery nor direct injection into target tissue haveproven successful. Intravascular delivery with the objective that thecells will find their way to where they are needed, has proven highlyinefficient. Direct injection into the site has also delivered poorresults, with an extremely high necrosis rate and low cell integrationrate.

The biocompatible scaffolds of the invention are useful in a broad rangeof in vivo and in vitro regenerative medicine and tissue engineering.Devices are designed and manufactured for a wide variety of injuries,diseases, conditions and cell therapies, and delivered to the treatmentlocation using surgical, endoscopic, endovascular, and other techniques.The devices degrade and resorb after the treatment is successfullycompleted or remain in place permantly or semi-permanently. Cells areseeded ex vivo into the scaffold with autologous or allogeneic cells.The devices are particularly useful in regenerating heart tissue(ischemia lesions and scarring), dermal tissue (scarring, ulcers, bums),CNS tissue (spinal cord injury, MS, ALS, dopamine shortage), and forskeletal-muscle system repairs (tendons, ligaments, discs,post-surgical, hernias)

A method for treating a patient in need of tissue regeneration,replacement or repair comprises the step implanting a scaffold in ornear the tissue in need of regeneration, repair or replacement. Thismethod for treating a patient in need of muscle repair involvesimplanting in the patient a biocompatible scaffold containing amacroporous, polymer matrix having at least one cell adhesion moleculeincorporated therein, a population of myoblast cells capable of muscleregeneration transplanted within the polymer matrix; and at least onecell growth inductive factor that prevents terminal differentiation ofthe transplanted cells in the polymer matrix and induces migration ofthe transplanted cells and their progeny out of the polymer matrix. Forexample, the cell growth inductive factor(s) is a combination of HGF andFGF2.

The devices are useful to treat acute and chronic tissue disease ordefects in humans as well as animals such as dogs, cats, horses, andother domesticated and wild animals. Conditions treated includeneuropathological disorders such as Amyotrophic Lateral Sclerosis (ALS),multiple sclerosis, polyneuropathy, multiple sclerosis (MS),Parkinson's, and epilepsy. Retinal diseases such as retinal degenerationand comeal injury (caustic) also can be treated with the devices. Thedevice can also be used to treat various heart and respiratory diseasessuch as myocardial infarction (MI), congestive heart failure (CHF),coronary artery disease (CAD), and cardiomyopathy or respiratorydiseases, e.g., chronic respiratory diseases (CRDs) or pulmonaryfibrosis, respectively.

Additionally, the device is used to treat bone and cartilagedefects/diseases such as periodontitis or a skull injury generally, butalso with craniotomy. Moreover, the device is implanted into or adjacentto neural tissues, e.g., to treat spinal chord injuries such as acrushed spinal cord. The device is used in other surgeries such as inmasectomies to heal and augment reconstruction of breast tissue.

Other uses include those that supply cells for treatment inhibition ofautoimmune diseases, such as Lupus, Mastocytosis, Scleroderma, andRheumatoid Arthritis. The device are also useful to supply cell fortreating blood disorders such as Sickle-cell Anemia or vacular disorderssuch as peripheral arterial disease (PAD), Peripheral Ischemia, ordiabetes.

Other diseases the device can be used to treat are gastrointestinal (GI)graft vs host, fenal failure, or Crohn's Disease. Additionally thedevice used for male infertility; for example, the device is implantedinto a testicle and functions as a surrogate spermatogonia to produce asteady stream of sperm cells. Skin diseases, injuries, or defects thatare treated with the device include skin burns and ulcers. Surgicaldefects such as those resulting from Caesarian section births and thoseresulting from cosmetic surgery are particularly amenable to treatmentusing flexible device scaffolds. Alternatively, the delivered orprogrammed/reprogrammed cells delivered from the device maintain tissueand organ structure and function (e.g., prevent age-related alterationsor deterioration).

The devices increase the efficacy of stem and transgenic cell therapies,and the devices are tailored to suit each clinical problem with theappropriate choice of scaffold composition, pore size, bioactivesubstance(s) and cell types. The device solves the major problem ofefficiently integrating therapeutic cells into target tissue. Physiciansplace the device near the site requiring therapy or regeneration, whereit delivers a flow of cells to the target site. Unlike traditionalscaffolds, th device exports cells after they have incubated, replicatedand matured inside the device. The device has shown 20×+ improvements inviable cell delivery and tissue re-growth for damaged skeletal muscle.By matching its design to the specific cell type biochemistry, thedevice causes an extended stream of matured cells to migrate into thetarget tissue.

The devices offer several advantages over other scaffold systems.Maximum therapeutic efficacy is achieved, because cells are delivered inprime condition at the right time in the right quantities directly tothe locus of disease or injury. Sustained delivery facilitates accretiveintegration of therapeutic cells into tissue at a desired location. Thedevices has been shown to be more efficient in viable cell delivery(110% for this device vs. 5% for the best alternative techniques). Thus,fewer cells are needed per treatment allowing successful therapies whichmight have failed at lower cell delivery rates. Lower cell numbers alsopermit autologous grafts, because fewer cells need to be harvested fromthe patient to be treated and 1Less time is required between harvest andgraft to proliferate cells in vitro. Since fewer cells are required,relative rare cells can be used. The devices also permit less expensiveallogeneic grafts. Other advantages include rapid determination of thetherapeutic benefit of any treatment and faster tissue growth andenhanced healing.

Vaccine Device

The biocompatible scaffolds are useful as delivery vehicles for cancervaccines. The cancer vaccine stimulates an endogenous immune responseagainst cancer cells. Currently produced vaccines predominantly activatethe humoral immune system (i.e., the antibody dependent immuneresponse). Other vaccines currently in development are focused onactivating the cell-mediated immune system including cytotoxic Tlymphocytes which are capable of killing tumor cells. Cancer vaccinesgenerally enhance the presentation of cancer antigens to both antigenpresenting cells (e.g., macrophages and dendritic cells) and/or to otherimmune cells such as T cells, B cells, and NK cells. Although cancervaccines may take one of several forms, their purpose is to delivercancer antigens and/or cancer associated antigens to antigen presentingcells (APC) in order to facilitate the endogenous processing of suchantigens by APC and the ultimate presentation of antigen presentation onthe cell surface in the context of MHC class I molecules. One form ofcancer vaccine is a whole cell vaccine which is a preparation of cancercells which have been removed from a subject, treated ex vivo and thenreintroduced as whole cells in the subject. These treatments optionallyinvolve cytokine exposure to activate the cells, genetic manipulation tooverexpress cytokines from the cells, or priming with tumor specificantigens or cocktails of antigens, and expansion in culture. Dendriticcell vaccines activate antigen presenting cells directly, and theirproliferation, activation and migration to lymph nodes is regulated byscaffold compositions to enhance their ability to elicit an immuneresponse. Types of cancers to be treated include central nervous system(CNS) cancers, CNS Germ Cell tumor, lung cancer, Leukemia, MultipleMyeloma, Renal Cancer, Malignant Glioma, Medulloblastoma, and Melanoma.

For the purpose of eliciting an antigen-specific immune response, ascaffold device is implanted into a mammal. The device is tailored toactivate immune cells and prime the cells with a specific antigenthereby enhancing immune defenses and destruction of undesired tissuesand targeted microorganisms such as bacterial or viral pathogens. Thedevice attracts appropriate immune cells, such as macrophages, T cells,B cells, NK cells, and dendritic cells, by containing and/or releasingsignaling substances such as GM-CSF. These signaling substances areincorporated in the scaffold composition in such a way as to controltheir release spatially and temporally using the same techniques used tointegrate other bioactive compounds in the scaffold composition.

Once the immune cells are inside the device, the device programs theimmune cells to attack or cause other aspects of the immune system toattack undesired tissues (e.g., cancer, adipose deposits, orvirus-infected or otherwise diseased cells) or microorganisms. Immunecell activation is accomplished by exposing the resident immune cells topreparations of target-specific compositions, e.g, ligands found on thesurface of the undesired tissues or organisms, such as cancer cellsurface markers, viral proteins, oligonucleatides, peptide sequences orother specific antigens. For example, useful cancer cell-specificantigens and other tissue or organism-specific proteins are listed inthe table below.

The device optionally contains multiple ligands or antigens in order tocreate a multivalent vaccine. The compositions are embedded in or coatedon the surface of one or more compartments of the scaffold compositionsuch that immune cells migrating through the device are exposed to thecompositions in their traverse through the device. Antigens or otherimmune stimulatory molecules are exposed or become exposed to the cellsas the scaffold composition degrades. The device may also containvaccine adjuvants that program the immune cells to recognize ligands andenhance antigen presentation. Exemplary vaccine adjuvants includechemokines/cytokines, CpG rich oligonucleotides. or antibodies that areexposed concurrently with target cell-specific antigens or ligands.

The device attracts immune cells to migrate into a scaffold where theyare educated in an antigen-specific manner and activated. The programmedimmune cells are then induced to egress towards lymph nodes in a numberof ways. The recruitment composition and deployment signal/composition,e.g., a lymph node migration inducing substance, is released in one ormore bursts, programmed by the method of incorporation and/or releasefrom the scaffold material, or controlled by the sequential degradationof scaffold compartments which contain the attractant. When a burstdissipates, the cells migrate away. Compartments containing repulsivesubstances are designed to degrade and release the repulsive substancein one or more bursts or steadily over time. Relative concentration ofthe repulsive substances cause the immune cells to migrate out of thedevice. Alternatively, cells which have been placed in or have migratedinto the device are programmed to release repulsive substances or tochange their own behavior. For example, localized gene therapy iscarried out by cell exposure to plasmid DNA attached to the scaffold.Useful repulsive substances include chemokines and cytokines.Alternatively, the device may cause immune cells to egress by degradingand releasing them.

Target disease states, stimulatory molecules and antigens useful invaccine device construction are listed below.

Bioactive factors to promote immune responses a. Interleukins: IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 IL-15, IL-18 etc. b.TNF-α c. IFN-γ d. IFN-α e. GM-CSF f. G-CSF g. Ftl-3 ligand h. MIP-3 β(CCL19) i. CCL21 j. M-CSF k. MIF l. CD40L m. CD3 n. ICAM o. Anti CTLA-4antibodies p. TGF-β q. CPG rich DNA or oligonucleotides r. Sugarmoieties associated with Bacteria: Lipopolysacharides (LPS) is anexample s. Fas ligand t. Trail u. Lymphotactin v. Mannan (M-FP) w. HeatShock Proteins (apg-2, Hsp70 and Hsp 90 are examples) Diseases andantigens - vaccination targets a. Cancer: antigens and their sources i.Tumor lysates extracted from biopsies ii. Irradiated tumor cells iii.Melanoma 1. MAGE series of antigens (MAGE-1 is an example) 2.MART-1/melana 3. Tyrosinase 4. ganglioside 5. gp100 6. GD-2 7.O-acetylated GD-3 8. GM-2 iv. Breast cancer 1. MUC-1 2. Sos1 3. Proteinkinase C-binding protein 4. Reverse trascriptase protein 5. AKAP protein6. VRK1 7. KIAA1735 8. T7-1, T11-3, T11-9 v. Other general and specificcancer antigens 1. Homo Sapiens telomerase ferment (hTRT) 2.Cytokeratin-19 (CYFRA21-1) 3. SQUAMOUS CELL CARCINOMA ANTIGEN 1(SCCA-1), (PROTEIN T4-A) 4. SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2)5. Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049) 6. MUCIN 1(TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHICEPITHELIAL MUCIN), (PEM), (PEMT), (EPISIALIN), (TUMOR-ASSOCIATEDEPITHELIAL MEMBRANE ANTIGEN), (EMA), (H23AG), (PEANUT-REACTIVE URINARYMUCIN), (PUM), (BREAST CARCINOMA- ASSOCIATED ANTIGEN DF3) 7. CTCL tumorantigen se1-1 8. CTCL tumor antigen se14-3 9. CTCL tumor antigen se20-410. CTCL tumor antigen se20-9 11. CTCL tumor antigen se33-1 12. CTCLtumor antigen se37-2 13. CTCL tumor antigen se57-1 14. CTCL tumorantigen se89-1 15. Prostate-specific membrane antigen 16. 5T4 oncofetaltrophoblast glycoprotein 17. Orf73 Kaposi's sarcoma-associatedherpesvirus 18. MAGE-C1 (cancer/testis antigen CT7) 19. MAGE-B1 ANTIGEN(MAGE-XP ANTIGEN) (DAM10) 20. MAGE-B2 ANTIGEN (DAM6) 21. MAGE-2 ANTIGEN22. MAGE-4a antigen 23. MAGE-4b antigen 24. Colon cancer antigenNY-CO-45 25. Lung cancer antigen NY-LU-12 variant A 26. Cancerassociated surface antigen 27. Adenocarcinoma antigen ART1 28.Paraneoplastic associated brain-testis- cancer antigen (onconeuronalantigen MA2; paraneoplastic neuronal antigen) 29. Neuro-oncologicalventral antigen 2 (NOVA2) 30. Hepatocellular carcinoma antigen gene 52031. TUMOR-ASSOCIATED ANTIGEN CO-029 32. Tumor-associated antigen MAGE-X233. Synovial sarcoma, X breakpoint 2 34. Squamous cell carcinoma antigenrecognized by T cell 35. Serologically defined colon cancer antigen 136. Serologically defined breast cancer antigen NY-BR-15 37.Serologically defined breast cancer antigen NY-BR-16 38. Chromogranin A;parathyroid secretory protein 1 39. DUPAN-2 40. CA 19-9 41. CA 72-4 42.CA 195 43. Carcinoembryonic antigen (CEA) b. AIDS (HIV associatedantigens) i. Gp120 ii. SIV229 iii. SIVE660 iv. SHIV89.6P v. E92 vi. HC1vii. OKM5 viii. FVIIIRAg ix. HLA-DR (Ia) antigens x. OKM1 xi. LFA-3 c.General infectious diseases and associated antigens i. Tuberculosis 1.Mycobacterium tuberculosis antigen 5 2. Mycobacterium tuberculosisantigen 85 3. ESAT-6 4. CFP-10 5. Rv3871 6. GLU-S ii. Malaria 1. CRA 2.RAP-2 3. MSP-2 4. AMA-1 iii. Possible mutant influenza and meningitisstrains d. Neuro protection - protect against neuro- logical diseases(e.g., Alzheimer's, Parkinsons, Prion disease) 1. Classes of self CNSantigens 2. human alpha-synuclein (Parkinson's) 3. beta amyloid plaques(Alzheimer's) e. Autoimmune Diseases (multiple sclerosis, Rheumatoidarthritis etc) i. Disease linked MHC antigens ii. Different classes ofSelf antigens iii. Insulin iv. Insulin peptide B9-23 v. glutamic acidvi. decarboxylase 65 (GAD 65) vii. HSP60 Disease linked T-cell receptor(TCR)

EXAMPLE 1 Designing Scaffolds to Enhance Transplanted Myoblasts Survivaland Myogenesis

Myoblast transplantation is currently limited by poor survival andintegration of cells into host musculature. Transplantation systems thatenhance the viability of the cells and induce their outward migration topopulate injured muscle enhances the success of this approach to muscleregeneration. Enriched populations of primary myoblasts were seeded ontodelivery vehicles formed from alginate, and the role of vehicle designand local growth factor delivery in cell survival and migration wereexamined. Only 5+/−2.5%, of cells seeded into nanoporous alginate gelssurvived for 24 hrs, and only 4+/−0.5% migrated out of the gels.Coupling cell adhesion peptides (e.g., G₄RGDSP) to the alginate prior togelling slightly increased the viability of cells within the scaffold to16+/−1.4%, and outward migration to 6+/−1%. However, processingpeptide-modified alginate gels to yield macroporous scaffolds, incombination with sustained delivery of HGF and FGF2 from the material,dramatically increased the viability of seeded cells over a 5 daytime-course, and increased outward migration to 110+/−12%. These dataindicate that long-term survival and migration of myoblasts placedwithin polymeric delivery vehicles is greatly increased by appropriatescaffold composition, architecture, and growth factor delivery. Thissystem is particularly useful in the regeneration of muscle tissue, andis broadly useful in the regeneration of other tissues.

The presence of bioactive compositions in or on a scaffold materialmaintains the viability of the cells for extended time periods whilesimultaneously encouraging outward migration of the cells to populatesurrounding host muscle fibers. Biodegradable polymer matrices thatco-deliver satellite cells and inductive molecules that signalendogenous cells to participate in muscle regeneration are specificallyuseful in this approach. The role of coupling cell adhesion ligands tothe matrix, material pore structure, and growth factor delivery from thematerial were studied in vitro. Alginate, a hydrophilic, biocompatiblepolysaccharide derived from seaweed, has carboxylic acid functionalgroups that allow covalent modification with cell adhesion peptides,allowing for the controlled presentation of signals that induce tissuedevelopment. In addition, controlling the molecular weight distributionof the polymer used to form gels allows one to regulate gel degradationand to increase the viability of alginate encapsulated cells. Takentogether, these properties make alginate hydrogels a useful modelmaterial for these studies. Finally, in addition to primary myoblasts, amyoblasts cells line (C2C12 cells) that produces characteristic muscleproteins, was used as a model system for the analysis of the expressionof myogenic proteins.

The following materials and methods were used to generate the datadescribed herein.

Alginate Modification

Low molecular weight (M_(w)=5.3×10⁴ g/mol, abbreviated as LMW) modifiedalginate, was produced by irradiating ultra pure MVG alginate powder(Pronova, Oslo Norway) with a cobalt-60 source for 4 hours at a γ-doseof 5.0 Mrad (Phoenix Lab, University of Michigan, Ann Arbor, USA). Highmolecular weight (MVG, Pronova, Mw=2.7×10⁵ g/mol,) alginate, ultra puregrade was also used to fabricate scaffolds. Both alginates were modifiedwith covalently conjugated oligopeptides with a sequence of G₄RGDSP(Commonwealth Biotechnology Inc.) at an average density of 3.4 mMpeptide/mole of alginate monomer, using carbodiimide chemistry known inthe art (e.g., Rowley, Madlambayan et al. 1999 Bio. Mat. Res.60:217-233; Rowley J. 2002 Bio. Mat. Res.20:45-53). 2% irradiatedalginate solutions were frozen and lyophilized until completely dry.Lyophilized alginate was added to MES buffer (Sigma) to yield a 1% w/vsolution and EDC, Sulfo-NHS and RGDSP peptide were added to thedissolved alginate and allowed to react for 20 hours. The reaction wasquenched with hydroxylamine and the solution was dialyzed withdecreasing concentrations of NaCl (7.5%, 6.25%, 5%, 3.75%, 2.5% 1.25%and 0%), over 3 days. The solution was purified via the addition ofactivated charcoal and subsequent sterile filtration. Sterile filteredalginate was frozen and lyophilized and stored at −20° C. Finally, themodified alginates were reconstituted in calcium-free DMEM (Invitrogen)to obtain 2% w/v solution (50% LMW; 50% MVG used in all experiments)prior to gelation. Reconstituted alginate was stored at 4° C.

Scaffold Fabrication

Three physical forms of scaffolds were prepared: nanoporous,microporous, and macroporous. To fabricate nanoporous alginatescaffolds, 5 ml non-modified or peptide modified alginate containingmyoblasts (10⁶ cell/ml) was crosslinked by adding 200 μl of CaSO₄ (0.41g CaSO₄/ml dd H₂O) (Aldrich), and the resulting solution was expressedinto molds (2×2×5 mm) constructed from polyvinylsulfoxane (PVS), (Kerr).The alginate was allowed to completely gel, and placed at 37° C. in highglucose DMEM. To form microporous (10-20 μm pores) scaffolds, alginatewas gelled in the absence of cells and then frozen at −120° C. Thefrozen scaffolds were lyophilized and stored at −4° C. until seeded withcells. To fabricate macroporous alginate scaffolds (400-500 μm diameteraligned pores), the alginate/calcium sulfate solution was expressed intothe PVS mold containing wire porogens (RMO othodontic wire, PO Box17085, Denver Colo.). A sterile glass plate was placed over the moldcontaining the alginate, and left undisturbed for 30 minutes. After thealginate has completely gelled (30 minutes), the alginate containing thewire porogens was frozen at −70° C. The frozen alginate gels werelyophilized overnight, wire porogens were carefully removed, and thedried scaffolds were stored at −20° C. until seeded with cells.

Myoblast Cultures

Myoblasts were derived from four week old C57BL/6 mice hindlimb skeletalmusculature. Under sterile conditions, the tibialis muscle of thehindlimb was surgically excised, finely minced and disassociated in0.02% Trypsin (GIBCO) and 2% Collagenase type 4 (WorthingtonBiochemical, Lakewood, N.J.) for 60 minutes at 37° C./5% CO₂ whileagitating on an orbital shaker. Disassociated cells were strainedthrough a 70 μm sieve, centrifuged at 1600 rpm for 5 minutes andresuspended in high glucose DMEM, with added pyruvate (GIBCO). Media wasfurther supplemented with 10% Fetal Bovine Serum (FBS) and 10%penicillin/streptomycin (P/S), (GIBCO) and this medium was used in allcell culture studies. Cells were plated and cultured at 37° C./5% CO₂for 72 hours before media change. After 72 hours in culture, the mediawas changed every 48 hours until cells were 80% confluent (about 7days). Cells were collected via centrifugation and overlaid on a Percollgradient (Amersham Biosciences, Uppsala, Sweden) in a 15 ml Falcon tube.The gradient consisted of 3 ml of 20% Percoll diluted in PBS (GIBCO), 3ml of 30% percoll diluted in DMEM (Invitrogen) and 3 ml of 35% Percolldiluted in Ham's F-12 (GIBCO). Cells were immediately centrifuged at1600 rpm for 20 minutes at 25° C. The cells from the 30% fraction werecollected and resuspended in high glucose DMEM.

Immunohistochemistry

To characterize myoblast cultures for the expression of myogenicproteins, Percoll purified primary myoblasts were plated on sterilecover slips overnight, and fixed in 0.2% paraformaldehyde for 20minutes. Coverslips were rinsed in phosphate buffered saline with 0.5%Triton-X (PBS-X), and incubated in Hoechst nuclear dye (1:1000).Coverslips were also incubated in an anti-desmin (1/100) monoclonalantibody (Chemicon, Temecula Calif.) followed by immunofluorescentsecondary antibody (1:1000), (FITC, Jackson Labs, West Grove, Pa.).After secondary antibody binding, coverslips were mounted on glassslides with aqueous mounting medium and sealed with clear nail polish.Slides were viewed with a conventional fluorescent light microscope(Nikon Eclipse E-800, Tokyo, Japan) or stored in total darkness forlater analysis. Images were captured utilizing NIH imaging software(National Institutes of Health, Bethesda, Md.), Spot digital camera andAdobe Photoshop.

Western Blot

Total cytoplasmic protein was collected by finely mincing modifiedalginate scaffolds containing primary myoblasts and placing theresultant solutions in 1.5 ml Eppendorf tubes. Fifty microliters ofpassive lysis buffer (Promega, Madison Wis.) was added directly to theminced scaffold and incubated at 37° C. for 10 minutes. The amount ofprotein in each sample was quantified by dilution of 1 μm of sample in200 μm protein assay reagent (BioRad), and the absorbance was measuredat 595 nm (Sunrise spectrometer).

All samples were denatured using standard SDS page protocols, loaded at25 μg total protein per well in 8% Tris Glycine polyacrylimide gels(Ready Gels, BioRad) and electrophoresed at 100 volts for 120 min(Laemmli, 1970). After electrophoresis, proteins were transferred toPDVF membranes (BioRad) at 100 volts for one hour utilizing the BioRadmini-blot. PDVF membranes were blocked in 1% bovine serum albumin (BSA)in Tris buffered saline with 0.1% Tween-20 (TBS-T) overnight at 4° C.After blocking, PDVF membranes were incubated in the appropriate primarymonoclonal antibody, either for desmin (1/100), myogenin (1/100), orMyoD (1/100), (Santa Cruz Biotechnologies, Calif.), for one hour at roomtemperature. Membranes were subsequently rinsed for 30 minutes in TBS-T(15 min×2), and incubated in horseradish peroxidase conjugated goatanti-mouse secondary antibody (BioRad), 1:1000 in TBS-T, for 1 hour.Membranes were rinsed in TBS-T for 30 min. (15 min×2) and proteins weredetected via chemiluminescent detection kit (ECL, Amersham) andvisualized by exposure to Hyperfilm ECL (Amersham).

Growth Factor Incorporation and Release Kinetics

To determine the release kinetics of HGF incorporated into modifiedbinary alginate scaffolds, a quantitative sandwich enzyme immunoassaytechnique (ELISA) was employed. Recombinant HGF protein (Santa CruzBiotechnologies, Calif.) was incorporated into alginate solutions priorto gelling (500 ng/ml), and gels were cast as previously described.After the gels had completely polymerized they were cut into 5 mmsquares and placed in 24 well plates, and 1 ml of PBS was added to eachwell. At various time points the PBS was removed and stored at 4° C. andfresh PBS added to the scaffolds. The PBS samples were measured fortotal HGF content via quantitative ELISA (Quantikine), and the resultswere compared to the initial HGF incorporated. To determine theincorporation efficiency and release kinetics of FGF2, 5 μci ofI¹²⁵-Bolton Hunter (PerkinElmer Life Sciences, Boston, Mass.) labeledFGF2 was incorporated into 2.5 ml of the alginate solution prior togelling. After gelling, gels were cut into squares (2×10×10 mm).Approximately 1 μci of labeled FGF2 was incorporated into each scaffold.Alginate scaffolds were placed in separate polypropylene tubescontaining 3 ml of phosphate buffered saline (PBS) and incubated at 37°C. At various time points the PBS was removed from the tubes, and freshPBS was added to the scaffolds. The release of FGF2 from the scaffoldswas determined by measuring the radioactivity present in the PBS removedfrom the scaffolds with a gamma counter (Beckman) and comparing theresult to the initial total I¹²⁵ FGF2 incorporated into the sample.

Migration and Viability

To determine the viability of primary myoblasts seeded in alginatescaffolds and to measure their ability to migrate out of the scaffolds,purified primary myoblasts were seeded in three dimensional alginatescaffolds (2×10⁶ cells/ml) in 24 well plates. A solution of cells inmedium (50 μl) was pipeted into each lyophilized scaffold; the mediumwas rapidly absorbed. The resulting viability and migration of myoblastsfrom alginate scaffolds, with both HGF and FGF2 (250 ng/scaffold)incorporation was subsequently measured by maintaining scaffolds inculture for various time points. To analyze the viability of cellswithin the scaffolds, the scaffolds were finely minced, treated with 50μl of trypsin and 50 μl of 5 mM EDTA for 5 minutes. Twenty μl ofdissolved alginate and suspended cells was then added to 20 μl of 4%Trypan Blue Solution (Sigma). The percent of viable cells was determinedvia trypan blue exclusion (dead cells appear blue due to their inabilityof exclude trypan blue from their nucleus), as viewed on a hemocytometerunder standard microscopic conditions (Nikon Eclipse E800, 20×). Tomeasure migration of myoblasts from the scaffolds, scaffolds were placedin new 24 well plates at various time points and the cells that hadcolonized the 24 well plates over the previous 24 hours were removed viatrypsinization and counted in a Coulter Counter (Beckman Corp.). Thetotal number of cells that migrated out of the scaffold was normalizedto the total number of cells initially seeded into the alginatescaffolds.

SEM Scaffold Characterization

The size and orientation of pores in alginate scaffolds were imagedutilizing a scanning electron microscope (ISI-DS 130, Topcon Techn. CA).All samples were dried and sputter coated (Desk II, Denton Vacum; NJ)prior to analysis.

Characterization of Myoblasts Isolated from Urine Hindlimb

Primary cells isolated from skeletal muscle were plated on coverslipsand analyzed by staining with a Hoescht nuclear specific stain, andimmunohistochemical staining for desmin. Analysis via light microscopyof random microscopic fields revealed that the initial isolation yieldeda heterogeneous population of cells, 75% of which expressed desmin (FIG.1A). To enrich the cell culture for myogenic cells, initial primary cellisolates were expanded in culture for 7 days, and subsequently purifiedvia Percoll density gradient fractionation. The resulting culturesconsisted of a 95% desmin positive population (FIG. 1B).

Role of Peptide Modification

The ability of myoblasts to both remain viable within and migrate fromscaffolds without cell adhesion peptides was first tested by seedingmyoblasts in scaffolds under the following three conditions: nanoporousalginate scaffolds, nanoporous scaffolds releasing HGF, and microporousscaffolds releasing HGF. Approximately ⅓ of incorporated HGF wasreleased in the first 10 hours, and a sustained release was observed forthe following time period (FIG. 2 a). The percentage of cells thatmaintained viability over the first 24 hours was less than 10% under allconditions (FIG. 2B). By 96 hours, a small percentage of viable cellscould only be measured in microporous scaffolds releasing HGF.Consistent with the cell loss was the minimal migration of myoblastsfrom the scaffolds (FIG. 2C). Over the first 24 hours, less than 5% ofthe total incorporated cells migrated from the scaffolds in any of theseconditions. This increased slightly by day two in the microporousscaffolds, but failed to further improve in any condition.

Covalent modification of alginate with adhesion oligopeptides prior toscaffold fabrication improved both the viability of cells, as well astheir outward migration (FIG. 3A-B). Myoblasts seeded in nanoporous,peptide-modified scaffolds demonstrated a two-fold increase in cellviability at 24 hours, as compared to cells in similar non-peptidemodified scaffolds. HGF release further increased viability to 40%, andcells seeded in peptide-modified microporous scaffolds with HGF releasedemonstrated a similar viability (FIG. 3A). However, the viabilitydecreased in all conditions over time. Similarly, cumulative migrationwas enhanced by peptide modification in concert with HGF release andmicroporosity. Peptide-modified nanoporous scaffolds led toapproximately 20% of seeded cells migrating out of the scaffolds (FIG.3B). Release of HGF by itself, and in combination with micropores led toan approximately 4-fold increase in cell migration over nonpeptide-modified nanoporous scaffolds (FIG. 3B). In those conditions,40% of the seeded cells migrated from the scaffolds, and were availableto fuse with host muscle fibers.

Macroporous Alginate and FGF2 Release

The effects of creating aligned pore channels (macroporous scaffolds;FIG. 4A), and FGF2 release (FIG. 4B) on the viability and outwardmigration of myoblasts were next examined. Creation of macroporessignificantly enhanced both the viability and migration of myoblasts(FIG. 5A-B), as compared to similar micro or nanoporous scaffolds.Release of HGF further increased both survival and migration ofmyoblasts. The percentage of viable cells under this condition was 63%at 24 hr, and an enhanced viability was maintained through the 96 hr ofthe experiment (FIG. 5A). Further, release of HGF and FGF2 together ledto the greatest level of outward migration (FIG. 5B).

Western blot analysis of cell lysates was performed to determine thedifferentiation state of cells within and migrating from thesescaffolds. Cells present in or migrating from scaffolds releasing HGFand FGF2 expressed high levels of MyoD (FIG. 6). Cells present inscaffolds without peptide modification nor HGF/FGF2 did not expresssignificant levels of MyoD. In contrast, neither cells within any of thescaffolds, nor those that migrated from the scaffolds expressedmyogenin, a transcription factor associated with end stagedifferentiation of myogenic cells (FIG. 6).

Enhanced Myocyte Survival and Migration to Target Tissues

The viability and ability of myoblasts to migrate from transplantedscaffolds was found to be strongly regulated by the presence of celladhesion ligands associated with the scaffold material, the porestructure, and release of growth factors from the material.

Incorporation of myoblasts into scaffolds lacking cell adhesion ligandsled to rapid and severe loss of viability, and cell migration wasminimal. However, modification of alginate with a G₄RGDSP peptideincreased cell viability and migration.

Alginate modified with a cell adhesion peptide(s) directed cells toadhere to the polymer, proliferate and in the case of myoblasts fuseinto myofibrils. Peptides that present motifs found in fibronectin,(e.g., RGD) are especially relevant to skeletal muscle engineeringbecause myoblast adhesion to fibronectin is associated with the earlyproliferative phase of myogenesis. The RGD peptide provided a signal formigration of myoblasts out of the scaffolds; however, additionalfeatures of the scaffold in combination with the RGD signal yielded moverobust migration. Alignment of macropores in the scaffolds led to ahigher cell survival at later time points, and most importantly, a veryefficient migration of cells out of the scaffolds. Macroporosity wasimportant for the migration of cells, and smooth muscle cells have beendemonstrated to grow most favorably on scaffolds with larger pores.

Incorporation of growth factors known, e.g., HGF and FGF in the case ofmyoblasts, significantly increased the viability and migration of seededmyoblasts under all variations of scaffold chemistry and architecture.FGF's were the first growth factors shown to have an effect on myogeniccells, and the FGF2 effect on myogenic cells is enhanced by the additionof HGF. In addition, myogenic repair in a muscle crush injury washindered by the injection of FGF2 antibodies. These data indicate animportant physiological role for FGF2, and a role for combined FGF2 andHGF signaling in skeletal muscle regeneration. However, injection ofFGF2 into skeletal muscle after injury did not enhance muscle repair,suggesting the importance of using this factor in the proper context. Inparticular, in the current approach to regenerate skeletal muscle,myogenic cells must be directed to bypass their normal tendency todifferentiate, and remain in a proliferative phase until a sufficientnumber of cells is attained to regenerate the tissue, and the cells havealso migrated from the scaffold. FGF2 is particularly useful inpreventing the premature differentiation of the transplanted cells,while the migration-inducing effects of HGF provide the latter function.

These results indicate that scaffolds for transplanted cells can beoptimized and designed for any phenotype to maintain cell viability andpromote migration out of the vehicle. Appropriate combinations ofscaffold architecture, adhesion ligands that maintain viability andallow migration, and growth factors that regulate phenotype are used incombination to obtain complex control over the fate of the transplantedcells.

Activation of Transplanted Cells for Muscle Regeneration

Macroporous alginate scaffolds were designed to serve as amicorenvironment for transplanted muscle progenitor cells (satellitecells) at a muscle laceration site. By releasing factors that promoteactivation and migration (hepatacyte growth factor and fibroblast growthfactor 2), but not terminal differentiation of satellite cells, cellscompetent to participate in regeneration of damaged host tissues arereleased in a sustained manner, effectively using the scaffold as aniche to sustain a pool of progenitor cells for regeneration of musclefibers.

In vivo studies were carried out using C57B1/6J mice in which thetibialis anterior muscle was completely lacerated at the midline of themuscle. Following laceration, the muscle ends were sutured togetherusing non-resorbable sutures, and scaffolds containing combinations ofgrowth factors and cells were placed on top of the injury. Muscleregeneration was assayed at 30 days.

Explanted tissues show largest area of new tissue formation when treatedwith scaffolds containing cells and both growth factors as quantified bymuscle mass and decrease in defect area.

Morphological analysis of regenerated muscle fibers showed increasedfiber diameter and an increase in the number of centrally located nucleiin animals treated with scaffolds containing growth factors and cells,as compared to all other sample types.

Transplantation of myoblasts derived from transgenic Rosa 26 mice thatcontain the β-galactosidease gene allows for observation ofincorporation of transplanted cells into regenerating host tissue.Transplanting cells on scaffolds containing growth factors increasesgrafting of transplanted cells into regenerating fibers as compared toinjection of cells without a scaffold or growth factors.

EXAMPLE 2 Niche Scaffolds Promote Muscle Regeneration Using TransplantedCells

Transplanting myoblasts within synthetic niches that maintain viability,prevent terminal differentiation, and promote outward migrationsignificantly enhances their repopulation and regeneration of damagedhost muscle. Myoblasts were expanded in culture, and delivered totibialis anterior muscle laceration sites in mice by direct injectioninto muscle, transplantation on a macroporous delivery vehicle releasingfactors that induce myoblast activation and migration (HGF and FGF), ortransplantation on materials lacking factor release. Controls includedthe implantation of blank scaffolds, and scaffolds releasing factorswithout cells. Injected cells in the absence of a scaffold demonstrateda limited repopulation of damaged muscle, and led to a slightimprovement in muscle regeneration. Delivery of cells on scaffolds thatdid not promote migration resulted in no improvement in muscleregeneration. Strikingly, delivery of cells on scaffolds which promotedmyoblast activation and migration led to extensive repopulation of hostmuscle tissue, increased the regeneration of muscle fibers, and led to ahigher overall mass of the injured muscle. This strategy for celltransplantation significantly enhance muscle regeneration fromtransplanted cells, and is broadly applicable to the various tissues andorgan systems.

The scaffolds described herein are not intended to guide tissueformation around the scaffold, but in contrast, maintain the viabilityof passenger cells while simultaneously encouraging and directing theiroutward migration to repopulate the surrounding host damaged tissue andto enhance its regeneration. The scaffold serves a function analogous tothe special tissue microenvironments, termed niches, that maintain thepotential of stem cell populations while allowing the daughter cells tomigrate and attain specialized functions distant to the niche. Thephysical and chemical aspects of the scaffold to successfully promotehost tissue repopulation and simultaneously prevent the prematureterminal differentiation of precursor cells.

Scaffolds were designed to promote myoblast survival, migration, andprevent terminal differentiation to enhance repopulation of injuredmuscle from transplanted myoblasts. As described above, HGF and FGF2release from macroporous scaffolds fabricated from RGD-presentingalginate polymers significantly enhanced the viability of satellitecells cultured in the scaffolds in vitro, and that HGF and FGF2 workedadditively to promote outward migration of the seeded cells whilepreventing terminal differentiation in the scaffold. An in vitro musclelaceration model was used to recapitulate injuries common in athletesand in trauma. This model is an accurate and reliable model of humaninjury. Some other mouse studies has been criticized in part due to theindirect relation of the models (irradiation, injection of cardiotoxin,or cryoinjury) to human injuries or disease. Further, to determine theparticipation of donor versus host myoblasts in muscle regeneration,donor myoblasts were obtained from Rosa26 mice to allow identificationby their over-expression of β-galactosidase.

Scaffold Preparation

Ultra pure MVG alginate powder (Pronova, Oslo Norway) was irradiatedwith a cobalt-60 source for 4 hours at a γ-dose of 5.0 Mrad (PhoenixLab, University of Michigan, Ann Arbor, USA to produce low molecularweight alginate (M_(w)=5.3×10⁴ g/mol). Alginates were further modifiedwith covalently conjugated oligopeptides with a sequence of G₄RGDSP(Commonwealth Biotechnology Inc.) at an average density of 3.4 mMpeptide/mole of alginate monomer, using carbodiimide chemistry. Highmolecular weight ultra-pure alginate (MVG, Pronova, M_(w)=2.7×10⁵ g/mol)was also covalently modified with this oligopeptide.

To fabricate alginate scaffolds that were highly porous, molds (2 mm×5mm×5 mm) were constructed from polyvinylsulfoxane (PVS) (Kerr). Porogenswere constructed from size 14 stainless steel orthodontic straight wirecut to 10 mm lengths. The orthodontic wire was aligned in two sets ofparallel rows 500 μm apart, sterilized and placed in the scaffold mold.A solution containing equal concentrations of irradiated low molecularweight (1%, w:v) and non-irradiated high molecular weight modifiedalginate (1%, w:v) was prepared in calcium free DMEM (Invitrogen). HGF(Santa Cruz Biotechnologies, CA.), and FGF2 (B&D) were added to thealginate solution (final concentrations 100 ng/ml). A calcium sulfateslurry (0.41 g CaSO₄/ml dd H₂O) (Aldrich), was added at a ratio of 40 μlCaSO₄/1 ml alginate and vigorously mixed. The resulting solution wasimmediately expressed into the PVS mold containing the wire porogens. Asterile glass plate was placed over the mold, and after the alginate hascompletely gelled (30 minutes), the gel containing the wire porogens wascarefully lifted from the PVS mold and placed in a 100 cm³ petri dish.To produce macroporous scaffolds with open, interconnected pores, thegels were cooled to −70° C., the wire porogens were carefully removed,and the gels were lyophilized and stored at −20° C. until needed.

Cell Culture and Seeding

Four month old B6.129S7-Gt(ROSA)26Sor/J (Jackson Laboratory, Bar HarborMe.) were sacrificed and the satellite cells were isolated from hindlimbs. Hind limb skeletal musculature was surgically excised, finelyminced and disassociated in 0.02% Trypsin (GIBCO) and 2% Collagenasetype 4 (Worthington Biochemical, Lakewood, N.J.) for 60 minutes at 37°C./5% CO₂ while agitating on an orbital shaker. Disassociated muscle wasstrained in a 70 μm sieve, centrifuged at 1600 rpm for 5 min. andresuspended in 10 ml high glucose DMEM, supplemented with pyruvate(GIBCO). Media was further supplemented with 10% fetal bovine serum and1% penicillin/streptomycin (GIBCO). Resuspended cells were plated in 75cm³ tissue culture flasks (Fisher), and HGF (50 ng/ml) and FGF2 (50ng/ml) were added to the medium. After seven days, cultures werepassaged and purified satellite cell suspensions were obtained viapercoll fractionation. Purified cultures were incubated for seven daysat 37° C. until 80% confluent and then were collected via trypsinizationand seeded at 10⁷ cells/ml onto modified open pore alginate scaffolds.

Surgical Procedure and Analysis

Four week old C57BL/6 mice were anesthetized via intra-peritonealinjection of ketamine (0.5 ml/kg) and xylazine (0.25 ml/kg). Bilateralincisions were made to expose the tibialis anterior muscle of bothhindlimbs. Once exposed, the muscle was completely lacerated at themidlength ventral-dorsally. The proximal ends of the lacerated musclewere then closed using a #4 black silk continuous suture, and scaffoldswere placed over the wound or myoblasts injected into the muscle. In allconditions utilizing myoblast transplantation, a total of 5×10⁵ cellswas delivered. The surgical site was closed with #4 black silkinterrupted suture and the surgical site was left undisturbed until themuscle was retrieved at 10 or 30 days.

Tibialis anterior muscle was excised and fixed in 4% paraformaldehydefor 2 hr and rinsed for 1 hr in PBS. Whole muscle was then incubatedovernight in β-galactosidase staining solution containing 25 μl/ml Xgalstock solution. The muscle was paraffin embedded, cut into serialsections (5 μm thick) and placed on glass slides for histologicalanalysis. Sections were deparaffinized through descending series of EtOHand rehydrated in H₂O and washed for 5 minutes in 3% H₂O₂ (Sigma) in PBSto quench any endogenous peroxidase activity. Sections were stained withGill's 3 hematoxylin (Sigma) and aqueous eosin solution (Sigma) tovisualize tissue morphology. Finally, serial sections were incubatedwith a monoclonal anti β-galactosidase antibody (1:1000), (Chemicon,Temecula Calif.) for one hour and then incubated with a HRP-conjugatedsecondary antibody (1:1000) (DakoCytomation, Carpinteria, Calif.).Samples were rinsed, and mounted with Permount (Fisher, Fairlawn, N.J.).

Defect size analysis was performed using Adobe Photoshop and Image ProPlus software. High powered (100×) images were obtained using a LeicaCTR 5000 light microscope and Open Lab software (Improvision). Sixsamples were analyzed for each condition. Areas of muscle defect wereidentified in hematoxylin and eosin (H&E) stained sections via theirlack of organized muscle fibers. Fiber size and nuclei number weredetermined via high powered microscopic analysis of ten random fields ofregenerating muscle fibers adjacent to the muscle defect. Only centrallylocated nuclei, a hallmark of regenerating muscle fibers, were countedin the quantification of number of nuclei.

Statistically significant differences were determined using two tailedstudents t-test. Statistical significance was defined by p<0.05. Alldata was plotted as the mean +/− standard deviation of the mean (SD).

Repopulation of Muscle Tissue with Transplanted Cells

The tibialis muscle of each mouse was lacerated and the laceration wassubsequently closed with suture. One of five conditions was used totreat the laceration site: 1) myoblasts were directly injected into themuscle at the laceration site, 2) blank scaffolds were placed over thelaceration, 3) scaffolds seeded with myoblasts were placed over thelaceration, 4) scaffolds releasing HGF and FGF2 (−cells) were placedover the laceration, and 5) scaffolds containing myoblasts and releasingHGF and FGF2 were placed. The implants were placed without the aid ofany adhesive or glue, and upon retrieval at 10 and 30 days, 80% of theimplants were in the same location as the day of surgery. The implantswere attached to the injury site and the overlying epidermis by fascialike tissue. A gross difference in the size of injured muscle treatedwith scaffolds containing growth factors and myoblasts, as compared toall other conditions, was observed at 30 days, as these muscles werelarger in every dimension than the other conditions tested (FIG. 7A-C).Quantification of the mass of these muscles revealed a statisticallysignificant 30% increase in mass, as compared to the other conditions(FIG. 7D). Gross observation also revealed that β-galactosidaseactivity, as indicated by lacZ staining, was noticeably more intense inmuscles treated with the scaffolds containing myoblasts and growthfactors (FIG. 7A) than in the other conditions in which myoblasts weretransplanted, indicating a greater repopulation of the native muscle bycells transplanted in this condition.

Analysis of tissue sections revealed a defect at 10 days that appearedlargely necrotic in all conditions (FIG. 8A-E). No normal muscle tissueappeared within the defect at this early time point. The defect wasfilled with cellular debris, blood and basophilic cells. There were nomyofibers that spanned the defect area. The muscle fibers that lined theborders of the defect were largely disorganized and contained centrallylocated nuclei. The muscle injury treated with a localized sustaineddelivery of growth factors alone had a larger remaining defect area thanany other condition at this time-point, although this difference wasonly statistically significant when compared to the injury treated withsustained delivery of both myoblasts and growth factors. When sectionsfrom muscle defects treated with myoblast transplantation were viewedunder high power magnification, there were no gross differences observedin the number of lacZ (+) cells present in the tissue at this time.

In contrast to the early results, the defects in the muscles treatedwith sustained localized delivery of myoblasts and growth factors werelargely resolved at 30 days (FIG. 8J). In many of these animals, theonly remaining defect was that caused by the closing suture. In additionthere were few areas of fat deposit and virtually no scar tissue at thistime point. The unresolved defect areas in the other experimentalconditions had also decreased in size (FIG. 8F-I), as compared to thedefect area at 10 days, but were still much larger than the cell/HGF andFGF2 delivery condition. In addition, scar tissue or fat deposits wereapparent in these other conditions. When the areas of unresolved defectswere quantified, there were no statistically significant differencesbetween the conditions at 10 days (FIG. 9A). However, at 30 days postinjury the defects in muscles treated with scaffolds delivering cellsand growth factors were significantly smaller than in any othercondition (FIG. 9B). A lesser reduction in defect size was also seen inmuscles treated with injected cells or scaffolds delivering HGF andFGF2.

To further analyze muscle regeneration, the mean width of regeneratedmyofibers and number of post mitotic centrally located nuclei per lengthof myofiber in the region proximal to the resolving muscle defects werequantified via high powered light microscopic analysis. The mean widthof regenerating fibers and density of centrally located nuclei werequalitatively greater in muscles treated with scaffold delivery of cellsand growth factors (FIG. 10B), as compared to scaffolds delivering onlygrowth factors (FIG. 10A), or any other experimental condition.Determination of the mean width of fibers 30 days post injury confirmedthat muscles treated with myoblasts in combination with growth factorsexhibited a 3 fold increase in fiber size as compared to the blankscaffolds, injected cells, or cells transplanted alone in scaffolds(FIG. 10C). The fiber width also increased in the experimental groupinvolving HGF and FGF2 delivery, but not as dramatically. In addition,the muscle fibers in the injury group treated with myoblasts and growthfactors via scaffold delivery contained 30% more centrally locatednuclei than any other conditions at 30 days post injury (FIG. 10D),indicating more fusion of myoblasts into the fibers, which supports thefinding that these fibers were larger in size.

Finally, immunostaining of tissue sections from the tibialis anteriormuscle 30 day post-injury revealed that the increases in the musclesize, fiber width, and fiber nuclei were accompanied by robustengraftment of transplanted myoblasts into host regenerating muscle,when cells were transplanted on scaffolds releasing HGF/FGF2 (FIG. 11A,C). A more limited number of engrafted donor cells were noted in thecondition using direct myoblast injection (FIG. 11B, D). No LacZ (+)cells were noted in the other experimental and control conditions.

Modulation of skeletal muscle regeneration, subsequent to injury, bymyoblast transplantation requires the survival of donor myoblasts andtheir stable incorporation into muscle fibers within the host tissue.Transplantation of myoblasts on scaffolds that promote their outwardmigration combines the advantages of host muscle fiber regenerationobtained with direct cell injection with the control over transplantedcell fate made possible with the use of cell-instructive scaffolds.Direct injection of myoblasts into injured muscles enhancesregeneration, as does localized delivery of HGF and FGF2 in combinationfrom a scaffold, but transplanting the cells from a scaffold thatsimultaneously delivers HGF and FGF2 dramatically enhanced theparticipation of transplanted cells in muscle regeneration and theoverall extent of regeneration.

Transplantation of myoblasts via direct injection, and delivery with ascaffold not releasing growth factors led to distinct outcomes in themodel system. Injection of myoblasts alone enhanced muscle regeneration,although to a modest extent. The injected cells participated in musclefiber formation, as evidenced by identification of Rosa26-derived cellsin the defect site, decreased mean defect size at 30 days, and increasedskeletal muscle fiber width. In contrast, transplantation of the samecell number on the scaffolds without growth factor release led to nodetectable changes in muscle regeneration, as compared to implantationof blank scaffolds at the defect site. Cell migration out of scaffoldsis low in the absence of the activating effects of HGF and FGF2 (20-30%of seeded cells migrate from scaffolds over 4 days in vitro) and thosescaffolds provide few cells to the surrounding tissue that canparticipate in regeneration compared to the HGF/FGF2 scaffolds.

Delivery of a combination of HGF and FGF2 from the scaffolds, in theabsence of transplanted cells, had a modest effect on muscleregeneration. The width of regenerating fibers was increased in thiscondition, as compared to blank scaffolds, and the number of centrallylocated nuclei in these fibers, a hallmark of regenerating myofibers wasincreased as well. These effects were consistent with the modestdecrease in defect area noted at 30 days. Other studies of local HGF andFGF2 delivery to sites of muscle regeneration have led to resultsdistinct from those reported herein. Local HGF delivery has beenpreviously documented to increase the number of activated myoblastswithin injured muscle, consistent with its role in activating satellitecells, but repeated presentation of HGF actually inhibited regeneration.Miller et al., 200 Am. J. Physiol. Cell. Physiol. 278: C174-181. Thehigh dose of HGF may have retarded the ability of host myoblasts towithdraw from the cell cycle and terminally differentiate. In addition,application of endogenous FGF2 had been previously reported to notenhance muscle regeneration. In contrast to those previous studies, thescaffolds described herein delivered small quantities of the factors(e.g., 5 ng), continually released the factors over an extended timeperiod, e.g., 3-10 days, delivered a combination of the two factorsrather than a single factor, and the type of muscle injury was alsodifferent from previous systems. The model system used to generate theforegoing data more closely resembles a human injury or muscle defectcompared to the earlier studies.

Transplanting myoblasts on a scaffold that released HGF and FGF2significantly enhanced muscle regeneration by every measure examined.The number of transplanted cells participating in muscle regeneration,as indicated by immunohistochemical staining for β-galactosidase,dramatically increased. The width of regenerating fibers wassignificantly enhanced, as was the number of centrally located nuclei inthe fibers, which are both consistent with an increased number ofmyoblasts participating in muscle formation. The enhanced regenerationled to almost complete resolution of the injury defect by 30 days, andto a significant recovery of muscle mass following the atrophy inducedby the injury. Cells placed in these growth factor releasing scaffoldsvery efficiently migrate out from the scaffolds in vitro (100% migrationin 4 days), and the growth factor release maintains the cells in anactivated, proliferating, but non-differentiated state (myoD positive,myogenin negative) in the scaffold. Prior to the invention, myoblastsinjected into muscle had poor survival due to the lack of an adhesionsubstrate and the inflammatory environment present in the injury.Transplantation of cells in scaffolds maintains the viability of thetransplanted cells, while protecting them from the inflammatoryenvironment. Activation of myoblasts by exposure to HGF and FGF2 alsoincreases their migration and proliferation, and thus enhances theirability to populate host musculature. The increase in muscle mass,muscle fiber size and the number of myonuclei per fiber, resemble thenormal regeneration of muscle tissue associated with healing of musclelacerations and other defects of muscular tissue (e.g., applicationsranging from hematopoietic system reconstitution to neuralregeneration).

EXAMPLE 3 Treatment Skin Wounds

In the context of a skin defect, the goals of the therapy are dictatedby the type of wound (e.g., acute bum, revision of scar, or chroniculcer) and size of the wound. In the case of a small chronic ulcer, theobjective is closure of the wound by regeneration of the dermis. Theepidermis regenerates via migration of host keratinocytes from theadjacent epidermis. For large wounds, keratinocytes, optimallyautologous, are provided by the device to promote regeneration of theepidermis. Cells are loaded into a scaffold material that is placeddirectly over the wound site, e.g., a scaffold structure in the form ofa bandage. The material provides a stream of appropriate cells topromote regeneration. For dermal regeneration, fibroblasts cells areused to seed the device, and these cells are either autologous (biopsytaken from another location and expanded before transplantation) orallogeneic. Advantages of autologous cells include a decreased risk ofdisease transmission, and immune acceptance of the cells. However, atime interval of several days to a few weeks would be required afterpatient biopsy to generate sufficient cells for treatment. Allogeneiccells allow immediate treatment of the patient from a stored bank ofcells, and significant reductions in therapy cost. Immunosuppressiveagents are optionally co-administered to reduce or prevent rejection ofthese cells by the patients immune system.

The device design includes one or a combination of the followingfeatures: 1. (physical properties) pores that would readily allow cellsto migrate out of the device into the underlying tissue; 2. (physicalproperties) a semipermeable external membrane designed to control fluidloss from the wound, prevent infection, and prevent cell migration outof the device away from the tissue; 3. (adhesion ligands) inclusion ofcell adhesion ligands to allow fibroblasts to migrate through and out ofthe material, e.g., RGD containing peptides; 4. (growth factors) localpresentation of FGF2 to induce fibroblast proliferation within thedevice; 5. (enzymes) the device is designed to allow for the rapidrelease into the wound of enzymes useful in debriding the wound; 6.(helper cells) if the individual was anticipated to have a limitedangiogenic response, endothelial cells or endothelial progenitors are beincluded in the device, and stimulated to repopulate the wound inconcert with the fibroblasts to promote vascularization.

This application utilizes materials with a relatively low elasticmodulus, e.g., 0.1-100, 1-100 kPa. Stiff materials would not besuitable, as such materials would not conform to a wound. Hydrogels orelastomeric polymers are useful in this device in order to conform tothe wound and provide control over fluid transport, prevent infection,and allow the physical contact required for cells to migrate out of thedevice into the wound. The hydrogel or other material also has adhesiveproperties. An adhesive surface permits contact to the wound so theremains fixed, even as the patient moves. Optionally, the device itselfis adhesive; alternatively, the device is fixed in place over the woundusing an adhesive composition such as a pharmaceutically acceptable tapeor glue. A semipermeable outer surface is provided by either using acomposite material (e.g., nonporous silicone sheet placed on outersurface of porous device) or processing the device to create anisotropicporosity.

EXAMPLE 4 Devices and Systems for Promoting Angiogenesis

Angiogenesis is a critical element in any tissue regeneration effort,and the temporally distinct signaling of vascular endothelial growthfactor (VEGF) is crucial in this process. Devices that containcompositions which promote angiogenesis together with endothelial cellsresulted in a synergistic angiogenic effect. The approach utilizes cellsthat play a role in the angiogenic process, e.g., endothelial progenitorcells and outgrowth endothelial cells (e.g., derived from cord blood orfrom peripheral blood samples).

An injectable alginate hydrogel was developed to provide spatialdistribution and temporal control of factors inducing neovascularizationof hypoxic tissues. The hindlimbs of C57BL/6J mice were made ischemic byfemoral artery and vein ligation, and the hydrogel containing growthfactors was directly injected into the ischemic muscle (bolus deliveryof VEGF was used as a control), and the in vivo release kinetics anddistribution of VEGF₁₂₁ and VEGF₁₆₅ were assessed using an ELISA ontissue samples. The gel led to complete return of tissue perfusion tonormal levels by day 28, whereas normal levels of perfusion were notachieved with bolus delivery of VEGF.

Several types of stem cells, including endothelial progenitor cells(EPC) cultivated from cord blood are useful in therapeutic angiogenesis.These cell-based therapies present several advantages over protein orgene-based therapies. Co-transplanting endothelial progenitor cells(EPC) and outgrowth endothelial cells (OEC) enhances vascularizationcompared to transplantation of each cell type alone. These cells aredelivered through the intramuscular injection as well as to othertissues in which vascularization is desired. Co-transplantation of EPCand OEC in a synthetic extracellular matrix device, which wasspecifically designed as a niche to support cell growth and migrationled to dramatically improved vascularization at an ischemic site.Microporous alginate-based hydrogels contained synthetic oligopeptidescontaining the Arg-Gly-Asp sequence (RGD peptides) and vascularendothelial growth factor (VEGF). The RGD peptide supports celladhesion, growth and migration in the gel matrix and the sustainedrelease of VEGF stimulates cell migration.

Hydrogels were prepared by cross-linking alginate molecules containingcovalently bound RGD peptides with calcium ions. VEGF was loaded in thegel matrix by mixing with alginate solution prior to cross-linking.Micro-sized pores in the gel matrix were induced by freezing the gel at−20° C. followed by lyophilization. Human microvascular endothelialcells were seeded into alginate scaffolds and placed into a collagen gelin a 24 well plate. After 3 days of culture, gel was degraded and cellnumber was quantified.

The mixture of EPC and OEC was loaded into the micropores andtransplanted to the ligation site. The femoral artery in the hind limbof SCID mice was ligated and the ends of the artery were tied off withsutures. The recovery of blood perfusion in the right hind limb wasevaluated using laser Doppler perfusion imaging (LDPI) system.

Human endothelial cells placed in matrices containing the RGD peptidemigrated out of the alginate gel scaffolds and populated the surfaces ofculture dishes in contact with the matrix, but their outward migrationwas significantly enhanced by the inclusion of VEGF in the matrix. Ahigher number of endothelial cells were localized in collagen andquantified at day 3 (FIG. 12) Transplanting EPC and OEC within syntheticmicroenvironments, including VEGF, completely recovered the bloodperfusion in the right hind limb within 6 weeks (FIG. 13 a). Incontrast, the bolus injection of cells led to limited recovery of bloodperfusion, and eventually the right hind limb was lost to necrosis (FIG.13 b). Transplanting both EPC and OEC within the gel matrix led to asuperior recovery of blood perfusion, as compared with transplantingeither EPC or OEC alone within the gel matrix (FIG. 13 c). FIG. 13 dfurther illustrates the recovery of blood perfusion in the animalstested. The device and cell niche system described above providestransplanted cells with the proper microenvironment that leads tosynergistic enhancement of vascularization in vivo.

EXAMPLE 5 Vaccine Devices that Regulate Cell Migration

Polymeric-based delivery systems were designed to regulate local in vivocellular migration. Cells of the body into which the device isadministered enter the device/scaffold, pick up an agent (e.g., a targetantigen or immune stimulatory molecule), and later emigrate to distantsites. These types of polymeric systems are especially useful in tissueand cellular engineering applications or vaccination protocols that seekto deliver molecules, such as peptides, proteins, oligonucleotides,siRNA or DNA to specific target cells in vivo that effectively modulatestheir function. The device recruits cells of the body into the scaffoldwhere the cells encounter agents that alter their function (e.g., thestate of differentiation or activation), and the modified cells leavethe implant site and have biological effects at diseased sites orelsewhere. Exit of the cells from the device is controlled by thecomposition, pore size, and or agents (e.g., cytokines) associated withthe device.

Delivery of GM-CSF from poly-lactide-co-glycolide matrices promoted invivo recruitment and infiltration of CD11c+ dendritic cells (DCs) in adose dependant manner (FIGS. 14A-C and 15A-B). Incorporation of afluorescent tag, fluoroscein, in the matrices permitted tracking of themigration of matrix of host DCs away from the scaffolds and into thedraining lymph nodes using flow cytommetry (FIG. 16A-C). GM-CSF deliveryenhanced the total number of DCs in the lymph nodes that were derivedfrom the implant site at days 14 and 28 after matrix implantation. Thesedata indicate that the device scaffold systems effectively both promotethe migration of cells into and out of a local site, in vivo, whilepicking up a bioactive agent, thereby modifying cell function such asimmune activation.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of inducing an endogenous immune response to a cancertarget, comprising administering to a mammal a device comprising ascaffold composition, said scaffold composition comprising open,interconnected macropores, a recruitment composition incorporated in oron said scaffold composition, and a target antigen composition whereinan endogenous antigen presenting cell is recruited into said devicewhere said cell encounters said target antigen and where said cellresides until a deployment signal induces egress of said cell via saidopen, interconnected macropores to a lymph node tissue outside of saiddevice, wherein the level of immune activation of said cell at egress isgreater than that prior to entering said device, and wherein saidrecruitment composition comprises a growth factor, cytokine, orchemokine that attracts bodily cells to said device, said target antigencomprises a cancer antigen, and said deployment signal comprises afactor that induces migration of said cell, thereby stimulating anendogenous immune response to said cancer target.
 2. The method of claim1, wherein said cytokine comprises GM-CSF.
 3. The method of claim 1,wherein said macropores are aligned.
 4. The method of claim 1, whereinsaid antigen presenting cell comprises a dendritic cell.
 5. The methodof claim 1, wherein said scaffold comprises a cancer-derived antigen. 6.The method of claim 5, wherein said cancer comprises a central nervoussystem (CNS) cancer, a CNS germ cell tumor, a lung cancer, leukemia,multiple myeloma, renal cancer, malignant glioma, medulloblastoma,melanoma, breast cancer, ovarian cancer, or prostate cancer.
 7. Themethod of claim 5, wherein said cancer-derived antigen is selected fromthe group consisting of MAGE series of antigens, MART-1/melana,Tyrosinase, ganglioside, gp100, GD-2, O-acetylated GD-3, GM-2, MUC-1,Sos1, Protein kinase C-binding protein, Reverse transcriptase protein,AKAP protein, VRK1, KIAA1735, T7-1, T11-3, T11-9, Homo Sapienstelomerase ferment (hTRT), Cytokeratin-19 (CYFRA21-1), SQUAMOUS CELLCARCINOMA ANTIGEN 1 (SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMAANTIGEN 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B) (KIAA0049),MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN),(POLYMORPHIC EPITHELIAL MUCIN),(PEM),(PEMT),(EPISIALIN),(TUMOR-ASSOCIATED EPITHELIAL MEMBRANE ANTIGEN),(EMA),(H23AG),(PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA-ASSOCIATEDANTIGEN DF3), CTCL tumor antigen se1-1, CTCL tumor antigen se14-3, CTCLtumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigense33-1, CTCL tumor antigen se37-2, CTCL tumor antigen se57-1, CTCL tumorantigen se89-1, Prostate-specific membrane antigen, 5T4 oncofetaltrophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus,MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGE-XP ANTIGEN)(DAM10), MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen,MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigenNY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinomaantigen ART1, Paraneoplastic associated brain-testis-cancer antigen(onconeuronal antigen MA2; paraneoplastic neuronal antigen),Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinomaantigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associatedantigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cellcarcinoma antigen recognized by T cell, Serologically defined coloncancer antigen 1, Serologically defined breast cancer antigen NY-BR-15,Serologically defined breast cancer antigen NY-BR-16, Chromogranin A,parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195, andCarcinoembryonic antigen (CEA).
 8. The method of claim 1, wherein saidscaffold comprises a tumor lysate.
 9. The method of claim 1, whereinsaid scaffold comprises irradiated tumor cells.
 10. The method of claim1, wherein said scaffold comprises a cancer cell-surface antigen. 11.The method of claim 1, wherein said scaffold comprises a viral orbacterial antigen.
 12. The method of claim 1, wherein said scaffoldfurther comprises an adjuvant.
 13. The method of claim 12, wherein saidadjuvant comprises a CpG rich oligonucleotide.
 14. The method of claim1, wherein said scaffold comprises RGD-modified alginate.
 15. The methodof claim 1, wherein said cell is immunologically activated at egresscompared to the level of immune activation prior to entering the device.16. The method of claim 1, wherein said cell is antigen primed at egresscompared to the level of priming prior to entering the device.
 17. Themethod of claim 1, wherein said scaffold comprises a hydrogel or porouspolymer, said scaffold comprising a polymer or co-polymer of polylacticacid, polyglycolic acid, PLGA, alginate, gelatin, collagen, agarose,poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone,polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide),poly(ethylene oxide), poly(allylamine), poly(acrylate),poly(4-aminomethylstyrene), pluronic polyol, polyoxamer, poly(uronicacid), poly(anhydride) or poly(vinylpyrrolidone).
 18. The method ofclaim 1, wherein said immune cell is selected from the group consistingof a dendritic cell, macrophage, T cell, B cell, and NK cell.
 19. Themethod of claim 1, wherein said deployment signal comprises a factorthat induces migration of said cell.
 20. The method of claim 1, whereinsaid deployment signal comprises a protein, peptide, or nucleic acid.21. The method of claim 1, wherein said device is implantedsubcutaneously into said mammal.
 22. The method of claim 1, wherein saiddevice is in the form of a bead, pellet, sheet, or disc.
 23. The methodof claim 17, wherein said porous polymer is produced by gas-foaming.